Like with other Donkey Kong boss levels, Donkey Kong has four lives and the amount of lives the player has is dependent on how many Mini-Marios were collected in the previous level. If the player selects this stage from the level select screen, they will begin with four lives.
Donkey Kong begins by tossing a Bob-omb up onto the highest conveyor belt, and the Bob-omb will begin to move down them. If a Bob-omb falls onto the second conveyor and is facing left, there is a small hole it can fit through to reach the player. In order to damage Donkey Kong, the player needs to press Direction Switches to keep the Bob-omb from going into the hole and to make it fall onto Donkey Kong before it blows up. However, there are several lasers that alternate around the Direction Switches and Donkey Kong has a Direction Switch of his own that he will use to keep the Bob-ombs away from him. Making four Bob-ombs fall onto Donkey Kong will defeat him.
A cinematic then plays, showing Mario chasing after Donkey Kong. Donkey Kong then realizes that his sack is empty, due to Mario having rescued all of the Mini-Marios. Mario begins to laugh at Donkey Kong, who is then joined in his laughter by three Toads and the Mini-Marios. Angry with this outcome, Donkey Kong grabs the Toads and climbs a tower. Mario gives chase and the next level begins, being another fight with Donkey Kong.