King K. Rool has stolen Donkey Kong's banana stash, and Donkey Kong needs your help to get them back. In search of K. Rool, the Kremling horde impedes your progress. Kremlings, crocodile-like creatures, include Kritters (they succumb to a simple jump on the head), Krushas (they are virtually indestructible), and others. There are also other enemies that block your path, such as beavers, snakes, vultures. Leading them are dastardly bosses, including Very Gnawty, Necky, Queen B., and Dumb Drum, and they all bar the way with their own special blend of villainy.

Donkey Kong is joined by Diddy Kong: these two primates differ significantly. For example, Donkey Kong is generally stronger; he can destroy most enemies with a single jump and can lift barrels straight over his head to throw them farther. On the other hand, Diddy is faster and not as strong as Donkey; he can run really fast and do cartwheels. Diddy carries barrels in front of him, creating a shield against frontal attacks.

The game is split into different worlds, which are split into different levels. Each level contains bonus areas, where one can collect bananas, medallions, and lives. At the end of each world is a boss, which is usually a larger version of a minor enemy.

The original Super Nintendo game was later released for Nintendo's handheld systems. The Game Boy Color version has an exclusive level (Necky Nutmare) and additional bonus games, Funky's Fishing and five Crosshair Cranky minigames. There was also the addition of collectible DK sticker packs scattered throughout the levels. These sticker packs could be printed out using the Game Boy Printer. The game also has two more difficulty options that become available after beating the game. Beating the game once lets the player turn off DK barrels and beating the game a second time will let the player turn off checkpoint barrels.

The Game Boy Advance version also had additional bonus games. It to has a version of Funky's Fishing but instead of the crosshair themed games, there is now Candy's Dance Studio, which hosts six dance games. There is also the additions of a time attack mode called DK Attack, a harder difficulty mode called Hero Mode (can only play as Diddy Kong and the game no longer offers Donkey Kong barrels and checkpoints throughout the levels), and the sticker book idea from the Gameboy Color version was replaced with collecting scrapbook pictures instead.