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Other T.U.F.F. agents (besides the main characters) made appearances throughout the show; however, most of these agents have no speaking roles yet.
- Dudley Puppy (Jerry Trainor) - The dimwitted and hyperactive white mixed-breed dog who wears nothing more than a black shirt and has a habit of chewing on his butt. Keswick's DNA analyzation shows that Dudley is the "perfect mix of every breed of dog known to man". He inherited the signature traits of each dog breed, such as a Bloodhound's sensitive nose, Greyhound's speed, German Shepherd's bravery and a "splash" of Billy Goat which cannot be explained, even by Keswick.
- Kitty Katswell (voiced by Grey DeLisle) - is T.U.F.F.'s number one agent as well as Dudley's partner and best friend. Skilled in various fighting styles and use of firearms, short tempered, and easily startled, this feline is a force to be reckoned with.
- Keswick (voiced by Jeff Bennett), works for the T.U.F.F headquarters as their primary inventor and scientist although whatever he invents is usually worthless due to the fact that it shoots acid or is too dangerous. Usually, when he speaks, he stutters and is similar to Jerry Lewis' on-screen persona as portrayed in The Nutty Professor. A running gag is that no one knows Keswick's species. It's shown that he has a pouch, gills, webbed feet, looks like a labradoodle and can lay an egg.
- The Chief (voiced by Daran Norris) - A tough but good-natured flea who runs T.U.F.F. headquarters. In "Internal Affairs", it's revealed that his real name is Herbert Dumbrowski, he has a bionic foot, and has short term memory loss.
Other T.U.F.F. agents
- Agent Nutz (also voiced by Jeff Bennett) - An agent who is a squirrel. He mostly says "stop following me".
- Agent Jumbo - An agent who is an elephant. She has a tendency to freak out whenever she sees a mouse, especially when Agent Rodentski appears.
- Agent Rodentski - mouse agent, in one episode used as camouflage by the Chameleon.
|1991–1997:||Doug • Rugrats • The Ren and Stimpy Show • Rocko's Modern Life • Aaahh!!! Real Monsters • Hey Arnold! • KaBlam! • The Angry Beavers|
|1998–2004:||Oh Yeah! Cartoons • CatDog • The Wild Thornberrys • SpongeBob SquarePants • Rocket Power • Pelswick • As Told by Ginger • The Fairly OddParents • Invader Zim • ChalkZone • The Adventures of Jimmy Neutron: Boy Genius • My Life as a Teenage Robot • All Grown Up! • Danny Phantom|
|2005–2009:||Avatar: The Last Airbender • Catscratch • The X's • Mr. Meaty • El Tigre • The Mighty B! • Back at the Barnyard • The Penguins of Madagascar • Fanboy and Chum Chum|
|2010–present:||Planet Sheen • T.U.F.F. Puppy • Kung Fu Panda: Legends of Awesomeness • The Legend of Korra • Teenage Mutant Ninja Turtles • Robot and Monster • Sanjay and Craig • Rabbids Invasion • Breadwinners|