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- Race: Monkey
- Color: Blue
- Diddy Kong, the eponymous monkey, is best known for his appearance in the first two Donkey Kong Country games. He appears on his own in the Nintendo 64 version of this game, without his Kong brethren.
- Race: Bear
- Color: Light Green
- Banjo is a honey bear from the Banjo-Kazooie series. This is the first appearance of Banjo in any game, even before the creation of the Banjo-Kazooie series (due to delays in the first game's eventual release date of 1998). Banjo's partner, Kazooie, is not seen in this game, but she is mentioned in the instruction manual and can also be heard yelling after Banjo's vehicle collides with an object.
- Race: Squirrel
- Color: White
- Conker is a squirrel and the main character from the Conker series. He is much more innocent in this game than he is in his later games, Conker's Bad Fur Day and its Xbox remake, Conker: Live & Reloaded.
- Race: Kritter
- Color: Orange
- Krunch is a Kremling (the antagonistic, crocodile-like species of the Donkey Kong Country series) who ends up wrapped up in the effort to kick Wizpig off the island. His clothing style was carried over to several of the Kremling enemies in the 1999 game, Donkey Kong 64.
- Race: Turtle
- Color: Light Blue
- Tiptup the turtle has high acceleration, low top speed, and good handling. He also makes cameo appearances in the games Banjo-Kazooie and Banjo-Tooie.
- Race: Tiger
- Color: Green
- Timber is a tiger cub who sports a blue baseball cap. His parents leave on vacation and have him watch Timber's Island in their absence. When Wizpig invades, Timber summons his friends to assist him in saving the island.
- Race: Badger
- Color: Yellow
- Bumper is a badger. He wears goggles on his head.
- Race: Mouse
- Color: Pink
- Pipsy is a mouse clad in a sailor suit, a polka-dotted bow, and gloves. In this version of the game, she is the only female character. In the Nintendo DS remake, Dixie Kong and Tiny Kong are the two additional females that replace Banjo and Conker (due to Rare, the company that created them, being bought by Microsoft).
- Race: Chicken
- Color: Red
- Drumstick the rooster is one of the greatest racers on Timber's Island. He was defeated by Wizpig and can be unlocked once he is saved.
- Race: Stopwatch
- Color: Gray
- T.T. is a living stopwatch who helped organize Time Trials. If players beat his records on every track, he becomes a playable character.