Murgatroid is first seen in a glass tank at Rob's lab. After Ratso makes a rude comment about Alberto, the Chihuahua retorts, "You stop that, you dirty rat, you!" Ratso then points out that rats are not dirty, just like snakes are not slimy. When Ratso asks Murgatroid if he's right, the snake replies in agreement, but hisses and spits his saliva all over Ratso. Disgusted, he advises Murgatroid to steer clear of words with too much "sssibilance."
Murgatroid is later present during the "Remember That Day" sequence and recalls the time when Rob (the Master) saved him from an aggressive mongoose in a neighbor's yard. Sometime later, Murgatroid and the other animals learn that Mack plans to ship them to Tartarus Laboratories to be used as test subjects. Sebastian, who had done some computer research earlier, noticed something on the screen before the computer was shut off due to a virus. When Murgatroid asks him where they're being sent, Sebastian explains the situation.
Ratso later leads Toaster and company to a recent disturbance that was coming from the basement. However, Mack shows up shortly after and prepares to ship the animals, but is stopped by Kirby (who was unable to fit inside the vent and was then given the duty to protect the animals). Murgatroid watches with delight as Kirby chases Mack around the lab, but the latter eventually manages to lock Kirby in a closet.
When Murgatroid and the other animals are eventually shipped by Mack, Toaster and friends chase down the truck. At one point, the truck goes over a few bumps in the road, shattering Murgatroid's glass tank. When Sebastian asks if he is okay, Murgatroid says he's fine, but just doesn't like having his cage rattled. Alberto then suggests that Murgatroid do something about it, to which the snake replies, "I jussst might do that."
Murgatroid then slithers over to Jim Bob and crawls up his leg, then his chest, and pops out from under his shirt, scaring him. Toaster and the gang eventually catch up with the truck, while Jim Bob gets out and complains that he can't stand snakes on him before he and Mack are arrested by the cops.
Back at the Master's lab, it is revealed by Chris that Murgatroid is going to take up residence in the reptile house at the zoo. The Master then gently scratches Murgatroid under the chin, much to the snake's pleasure.
Murgatroid is last seen in his new habitat at the zoo during the "Hang in There, Kid" sequence with a female snake, which he presumably mated, and they are revealed to have a baby snake of their own. He also appears briefly on Wittgenstein's computer screen with all the other protagonists of the film.
- He is the first Disney snake character who is a protagonist, the second being Larry from The Wild, the third being Juju from The Princess and the Frog.
- In real life, some snakes eat rats as part of their diet. Ironically, Murgatroid did not attempt to eat Ratso, even after the latter insulted him.