Mario, Luigi, Toad and Princess Toadstool, after some traveling, arrive in an unnamed town in the Swiss Cheese Alps, the villagers of which they plan to rescue from a mad scientist named Dr. Koopenstein, with Mario deducing that he is King Koopa. While in the village, Toadstool is greeted by the mayor, who gives her the key to the village; as this happens, Mario whispers to Luigi that he wonders if the key fits the village pizza parlor. As Toadstool thanks the mayor, who then says it is an honor for her, Mario, Luigi and Toad to have come to defeat Koopenstein, Mouser peeks around the corner before deciding to leave to tell Koopenstein of Toadstool, Mario, Luigi and Toad's presence. As Mouser leaves, Toadstool tells all the present villagers that she and her cohorts will try their best to defeat Dr. Koopenstein.
Nearby, at Koopenstein's tower, the doctor is in the midst of saying to himself that all he needs to complete his Robot Monster Troopa is a brain, and begins to laugh to himself. As Koopenstein snickers, Mouser enters his laboratory, calling out to him; seeing Mouser enter the laboratory, Koopenstein says aloud that if Mouser weren't so stupid, he could use his brain to power the Troopa, before going to ask Mouser what he wants. Answering Koopenstein, Mouser tells him that Princess Toadstool, Mario, Luigi and Toad are in the village, news that gives Koopenstein the idea of using both Mario and Luigi's brains to power the Troopa, believing that individually, the brothers' brains are too dim for use in the Troopa. After congratulating Mouser on informing him of Mario, Luigi, Toad and Toadstool's arrival, Koopenstein proceeds to threaten him, saying that if he doesn't procure Mario and Luigi's brains, he will feed him to the Troopa, a prospect which makes Mouser shiver in fear.
Outside Koopenstein's tower, Mario, as he climbs the stairs to the tower, says that he, Luigi, the Princess and Toad will defeat Koopenstein and be back before dinner, a rant which Luigi responds to by saying that first, they need to get into Koopenstein's tower. Hearing Luigi say this, Mario approaches the door of the tower and yells for whoever is there to open it up, saying that he, Luigi, Toad and the Princess have come to fix the plumbing. Hearing Mario, a Shyguy opens the wicket gate of the door and, upon seeing Mario, quickly slams it shut. Once the Shyguy leaves, Toad tells Mario that his plan caused the tower's inhabitants to know that the four heroes were there; as Toad makes this complaint, Mario asks him if he has a better idea, which Toad says he does, saying that he will climb the wall of the tower, sneak in, and let Mario, Luigi and Toadstool into the tower.
Having begun to escalate the wall, Toad, who is having a difficult time climbing, berates himself on having thought of such a hard to-do plan and for having a big mouth. Watching as Toad scales the tower wall, nearing the battlements, Mario yells for him to hurry, a statement which causes Princess Toadstool to ask Mario if he ever thinks about anything other than food. When Mario asks what else there is to think about, Mouser appears with a brigade of Shyguys (one of whom is riding an Autobomb), and responds "brain surgery," adding that he is going to learn about that the hard way. Oblivious to what is occurring below him, Toad continues to climb the tower wall, eventually reaching a battlement, where he finds a trio of Shyguys, an Autobomb and two potted Piranha Plants waiting for him.
In his laboratory, Dr. Koopenstein is working on a machine, when Mouser appears and announces his presence, as well as the presence of Mario, Luigi, and Toadstool, who are in shackles. Congratulating Mouser on a job well done once more, Koopenstein exclaims that he will be able to finish his brain transfer experiment tonight, before letting out a fit of maniacal laughter. On the battlement, Toad, who finds himself surrounded by several Shyguys, says that maybe a coin should be flipped; if it lands on heads, he keeps climbing and if it lands on tails, the Shyguys let him go, but either way the Shyguys can keep the coin. As a Shyguy carrying handcuffs advances on him, Toad tosses his coin into the air, which distracts the Shyguys. With the Shyguys preoccupied, Toad grabs a nearby potted Piranha Plant and, after jumping on a nearby Autobomb, has the plant light its fuse with a fireball. Dropping the Piranha Plant, Toad announces to the Shyguys that they lose, before jumping away just as the Autobomb launches an explosive right at the Shyguys.
In his laboratory, Dr. Koopenstein is in the midst of telling Mario and Luigi, who are strapped to a chair, that he is going to measure their collective brain power, and also tells them to think of the smartest person they know, him. Pulling a lever, Koopenstein activates his brain scanner, which reveals to him that Mario and Luigi aren't too bright, with Koopenstein exclaiming that a potted plant scores higher than they do. Outside, Toad, who is climbing the tower wall again, comments that the Shyguys following him must have velcro on their feet, before looking down and seeing three Shyguys literally walking up the tower wall after him. Eventually reaching a window, Toad looks in it to see Koopenstein yelling that no one can be as stupid as Mario and Luigi; Mario retaliates by saying that Dr. Koopenstein is that stupid if he thinks he and Luigi will give their brains to him. Hearing all this, Toad climbs to the top of the tower and reaches a skylight.
Inside the laboratory, Toadstool, after knocking Mouser aside, tells Koopenstein to leave Mario and Luigi alone. After Toadstool says this, Koopenstein sarcastically asks if Toad will help her; as if on cue, Toad suddenly leaps through the skylight and lands on Koopenstein's head, knocking him to the floor. As Mouser dives for cover, the Princess and Toad free Mario and Luigi from their bonds. Getting off the ground and yelling, Koopenstein attempts to tackle Luigi, only to miss him and crash into Mouser instead, knocking him away. As Mouser is flung away and starts yelling, Koopenstein is sent sprawling and crashes into his own brain scanning chair. Before he can get up, Koopenstein is electrocuted by his brain scanner, which sends out flashes of light. Blinded by the light, Mario, Luigi, Toad and the Princess finally open their eyes and see that Dr. Koopenstein has undergone a horrific transformation into a monster, which Mario dubs the Koopenstein Monster. As Mario yells for Luigi, Toad and Toadstool to run, the transformed Koopenstein lunges forward and, after knocking Mario, Luigi, and Toad away, grabs the Princess and crashes through a nearby door, with Mouser pursuing him. Getting off the ground, Mario, Luigi and Toad run outside, just in time to see Koopenstein carry Toadstool down the stairs leading to the tower, with Mouser running after the two.
While sitting on the steps leading to Koopenstein's tower, Mario begins to bemoan the loss of Princess Toadstool, saying to Luigi and Toad that Koopenstein could be anywhere. Just afterward, the mayor of the village appears and frantically tells Mario, Luigi and Toad that Koopenstein is attacking the village. Quickly rushing to the village, Mario, Luigi, Toad and the mayor find Koopenstein in the middle of terrorizing villagers and destroying property. As Koopenstein lets out a roar, Mario tells Luigi to get out his plunger, which Luigi says he left at home before trying to run away. Stopping Luigi from escaping, Mario tells him to forget his plunger, telling him to simply grab some Bomb Plants instead.
As Koopenstein chases a Mushroom child, Mario, Luigi and Toad leap in front of him and begin to taunt, which causes Koopenstein to grab Mario and throw him at a nearby clock tower, the hands of which Mario grabs to stop from falling. As Koopenstein charges towards Luigi, Toad steps in front of him and thrusts a Bomb Plant into Koopenstein before stepping away. After the Bomb Plant explodes in Koopenstein's claws, Luigi and Toad begin to celebrate their supposed victory over the Koopenstein Monster, only to see the creature unharmed by the Bomb Plant once the smoke caused by it clears. As Toad and Luigi stand dumbstruck, Koopenstein begins to walk towards them.
Seeing Luigi and Toad in peril, Mario begins to taunt the Koopenstein Monster, which causes it to begin to advance towards the base of the clock tower. As he nears the clock tower, Koopenstein is hit mesmerized by a loud cuckoo bird that has popped out of the tower. Angered by the cuckoo bird blaring in his ears, Koopenstein grabs the base of the clock tower and begins to shake, succeeding in tearing it out of the ground. With the clock tower in its clutches, the Koopenstein Monster proceeds to throw it through the air, with Mario still attached to it; luckily, Mario lands safely in a field filled with cows, but is knocked unconscious by the cuckoo bird in the clock tower, which lands nearby, taunting him. In the village, Luigi and Toad are being chased by Koopenstein, who manages to grab them and, letting out a sinister laugh, hurls them away.
Sent soaring away, Luigi and Toad land in the same field Mario is in; finding Mario unconscious on the ground, Luigi begins trying to wake him up, to no avail. Deciding to wake Mario up himself, Toad goes over to the clock tower and proceeds to kick it, causing the cuckoo bird to pop out and make enough noise to wake Mario up. Coming to his senses, Mario asks Luigi and Toad what happened, and gets the simple response from Toad that they lost the fight against the Koopenstein Monster. Up in the Swiss Cheese Alps, Koopenstein lumbers towards a cave, which Mouser exits. Greeting Koopenstein, Mouser tells him that he stopped Toadstool from escaping, which Koopenstein responds to by emitting several unintelligible grumbles. Entering the cave alongside Mouser, Toadstool yells at the Koopenstein Monster, demanding it to let her go, but Koopenstein quickly silences Toadstool by growling menacingly at her, which Mouser translates as meaning that Toadstool will never get away. In Koopenstein's laboratory, Toad is asking whether Mario's plan is going to work, which Mario responds to by saying that if it doesn't, everyone will be in big trouble. Taking out a remote control with a joystick on it, Mario begins to move around the joystick, which activates the nearby Robot Monster Troopa, scaring Toad, who hops into Luigi's arms.
In the village, Mario, who is riding the Robot Monster Troopa, is greeting a cheering crowd of villagers, as Luigi and Toad follow behind him, carrying Bomb Plants. Riding the Troopa out of the village, Mario announces that he plans to follow the Koopenstein Monster's footsteps to the creature’s lair, where he will defeat it. Outside Koopenstein's cave, Mouser is keeping watch over the area, when he sees, to his surprise, Mario, Luigi, Toad and the Troopa rapidly approaching. Running into Koopenstein's cave, where the monster is in the middle of menacing Princess Toadstool, Mouser informs Koopenstein that Mario, Luigi, Toad and the Troopa are coming. After Mouser tells him this, Koopenstein begins to leave the cave, only to be stopped by the Princess, who begs Koopenstein not to hurt Mario, Luigi or Toad. Ignoring Toadstool, Koopenstein leaves while Mouser holds her back, telling her not to interfere in his or Koopenstein's fun.
Seeing the Koopenstein Monster, Mario has the Robot Monster Troopa begin to battle it; as the Troopa and Koopenstein struggle against one another, the heroes and Mouser watch. Gaining the upperhand against Koopenstein, the Troopa manages to lift him above his head and throw him away. Getting off the ground, an angry Koopenstein rushes forward and shoves the Troopa to the ground; getting up, the Troopa attacks Koopenstein and the two begin to struggle again. Seeing that Mario has the Troopa's remote, Mouser rushes forward and, knocking the Bomb Plants Luigi and Toad were carrying away, grabs the remote control from Mario and begins to run away. Before he can get far, Mouser is tackled to the ground and sent skidding by Toad, and eventually stops behind the Troopa, who accidentally steps on its remote; with the remote destroyed, it releases an electrical surge that travels through the Troopa and into the Koopenstein Monster. As electricity flies through the air, Mouser, Koopenstein and the Troopa are sent tumbling off a nearby cliff and into a river.
As Toad, Luigi, Mario and Princess Toadstool look down below, Mouser and Koopenstein, who has been returned to normal, surface from the river. Unable to remember his tenure as the Koopenstein Monster, the doctor demands to know what has happened, and Mouser proceeds to tell him the long story, warning him in advance that he'll hate the ending.
In the village, where the clock tower has been restored, the mayor and a cheering crowd are thanking Mario, Luigi, Toad and the Princess for stopping Koopenstein; carrying a covered tray, the village mayor presents Mario with his reward, a pizza covered in hole-filled cheese. Seeing the holes in the pizza's cheese, Mario loudly asks whose been eating his pizza, only for the Princess to answer him saying that the holes in the cheese are normal, as it is Swiss cheese. Not satisfied, Mario asks about the key to the village, wanting to find a pizza parlor where he can show the villagers how to make pizza right, Mario walks out of the scene, as the Mayor, Luigi, the Princess, and Toad laugh.
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