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- Dudley Puppy - agent
- Kitty Katswell - agent
- Keswick - scientist
- The Chief - leader
- Agent Amoeba - agent
- Agent Ant - agent
- Agent Anteater - agent
- Agent Bacon - agent
- Agent Big Bad Wolf - agent
- Bobby - intern
- Agent Bossy - agent
- Agent Bull - agent
- Agent Deer - agent
- Agent Goldfish - agent
- Agent Groundhog - agent
- Agent Guinea Pig - agent
- Agent Hammerhead - agent
- Agent Jumbo - agent
- Agent Kid - agent
- Agent Moby - agent
- Agent Mouse - agent
- Agent Nutz - agent
- Agent Rodentski - field agent
- Agent Scarecrow - agent
- Agent Seabiscuit - agent
- Agent Sea Cucumber - agent
- Agent Skunk - agent
- Agent Snake - agent
- Agent Three Little Pigs - agent
- George Looney - janitor
- Jack Rabbit - former agent (became a villain in The Wrong Stuff)
- KIL-R - agent
- Nocturnal T.U.F.F. Agents - agents
- Percival - intern (formerly worked for The Stink Bug)
- R.I.T.A. - former agent (became a villain in Toast of T.U.F.F.)
- Scary Jimmy - janitor
- Sally Mander - secretary
- Tammy - secretary
- T.U.F.F. Troopers - agents
- Verminious Snaptrap - former agent (became an agent and then went back to being a villain in Snap Dad)
- The design of the T.U.F.F. Headquarters building is possibly a parody of the Titans Tower, from Teen Titans, or the tower from the episode The Architect, from Generator Rex.
- The agency's acronym is a pun on the word "tough".
- T.U.F.F. has a similarity to the S.P.D. headquarters from Tokusou Sentai Dekaranger & Power Rangers SPD.
- T.U.F.F. has teleportation tubes that go to Petsburg and the North Pole seen in both Mission: Really Big Mission and A Doomed Christmas.
- The animal picture on the T.U.F.F. badge is based of the animal the agent is.
- It is revealed that the doors aren't locked at T.U.F.F. as revealed in Freaky Spy Day.
- For a secret agency, that has "undercover" in the acronym, T.U.F.F.'s operations are never kept secret.
|Turbo Undercover Fighting Force|
| Dudley Puppy • Kitty Katswell • Keswick • The Chief |
Other Agents: Agent Amoeba • Agent Bacon • Bobby • Agent Bossy • Agent Bull • George Looney • Agent Goldfish • Agent Groundhog • Agent Guinea Pig • Agent Hammerhead • Agent Jumbo • Agent Kid • KIL-R • Agent Moby • Nocturnal T.U.F.F. Agents • Agent Nutz • Percival • Agent Rodentski • Agent Scarecrow • Agent Seabiscuit • Agent Sea Cucumber • Agent Skunk • Scary Jimmy • Sally Mander • Tammy • T.U.F.F. Troopers