Game: Once an ordinary earthworm from somewhere in Texas, Jim was mutated into a new form of life when an ultra high tech indestructible super space cyber suit (more commonly called just "the Super Suit") fell from space and landed atop him. Narrowly evading the bounty hunter Psycrow, Jim learned of the existence of the evil Queen Slug-for-a-Butt and her beautiful sister Princess Whats-Her-Name, and set out to see the princess.

Cartoon: As in the game, Jim was created when Psycrow lost the Super Suit and it landed on a Texan earthworm. Unlike the game, Jim instead set out to become a superhero, for reasons unknown. Flamboyant, naive, simple-minded and at least a little crazy, Jim is nevertheless one of the best heroes in the known universe.

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• American Accents: In the game, Jim has a slight but undeniable Texan Drawl accent. The cartoon drops this in favor of a hammy superhero-esque dialect.
• BFG: Subverted. Jim's primary gun is a small handheld ray-gun. It still packs a massive amount of firepower, and it's a rapid-fire weapon.
• Clothes Make the Superman: Jim is only sentient because of the energy from his suit mutating him from being an ordinary annelid. Although the boost to size and intellect is permanent, all of his strength, firepower and physical abilities are due to his suit.
• Heroic Sociopath: Jim's plan A boils down to "blast away randomly at any viable target while laughing maniacally".
  Jim: EAT DIRT, EVERYONE IN THE VICINITY!!!!
• Idiot Hero: Jim in the cartoon zigzags between this, occasionally coming up with some clever ideas to defeat his enemies, but generally being rather dense.
• Large Ham: In the cartoon.
• Psychopathic Manchild: In the cartoons, Jim's interests include watching a kid's show ("The Mr. Bunnybutt Show"), reading a funny animals popup book, firing his gun at anything that catches his attention, and pummeling people senseless.
• Small Name, Big Ego: The cartoon zigzags on this. Jim is crazy, egotistical and full of himself, but, more often then not, when the chips are down, he proves himself to be genuinely competent.

Game: A notorious mercenary and bounty hunter, feared for his determination, Psycrow was commissioned by Queen Slug-for-a-Butt to bring her the Super Suit newly constructed by Professor Monkey-for-a-Head. But on the way, he was attacked by an agent deployed from the Phlegm Galaxy by Major Mucus, who stole the Suit. Psycrow destroyed the agent's ship, but the suit plunged to the planet Earth, creating Earthworm Jim. Determined not to fail his mission, Psycrow stalked Jim all the way to the planet Insectica, striving to ambush Jim on each leg of his journey between planets.

Cartoon: A nefarious space villain, Psycrow roams the galaxy in search of plunder and excitement. Conquest, destruction, thievery, these are all part of a day's work for a Scourge of the Spaceways. He's not above hiring himself out as a mercenary or assassin, but he's quite comfortable working for himself.

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• American Accents: The cartoon gives Psycrow a "Joisey" accent.
• Arch-Enemy: In the games.
• Abnormal Ammo: In the games, Psycrow's primary weapon is a gun that fires giant fish hooks.
• Card-Carrying Villain: While nearly all of the Rogues Gallery display this trait in the cartoon, Psycrow seems to have the most devout professional life regarding it, several of his intervals being mock-commercials for villanous societies or victims of superheroism.
• Determinator: Psycrow doesn't give up easily.
• The Dragon: To Queen Slug-For-A-Butt on frequent occasions.
• Spell My Name with an S: Is it Psycrow or Psy-Crow?

Game: Immortal incarnation of evil, this vile cat seeks to eliminate all that is good and wholesome in the universe, forever plotting and devising new methods of tormenting and corrupting from his nightmarish homeworld of Heck. His twin brother Flagitious runs the Circus of Scars, and during the summer the two switch jobs.

Cartoon: Depraved ruler of Planet Heck, the source of all true evil, this mad feline yearns to destroy the universe.

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• Cats are Mean: The core precept behind Evil the Cat.
• Didn't Think This Through: In the cartoon, when questioned on what he plans to do in the aftermath of succeeding in destroying the entire universe, he admits he had never really thought about it. Doesn't stop him, though.
• Omnicidal Maniac: His cartoon version is obsessed with destroying the universe.
• Planet Heck: Rules the Trope Namer.
• Satan: In the games, he is basically this.
• Wicked Cultured: In the cartoons he has the most hellish and destructively evil ambitions of the Rouges Gallery, spaced in between the odd movie night and romantic affair of course.
  Evil: I'm just watching TV.

Game: Upon a world that is almost entirely water and subaquatic mountains, Jim encounters Bob, a small fish with incredibly high intelligence. Always more intelligent then the other fish surrounding him, Bob finally dared to approach the surface, where he found the limited landmasses housed dimwitted but incredibly powerful humanoid felines. Awestruck by Bob's cowardly screams, they took him in as their god, giving him a fishbowl so that he could be carried around and guide them. Enslaving the stupid brutes, Bob used his intellect to help them construct a labyrinthine underwater city. Ambition flooding him, Bob desires to conquer the universe — but what could a fish in a bowl do to achieve that goal? Jim's Super Suit is a way to compensate for his unimposing physique, and so he yearns to claim the suit for himself.

Cartoon: Fundamentally identical to the games, Bob in the cartoon series is depicted with a bombastic, evangelical personality. The planet is also renamed La Planeta de Agua (arriba!).

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• American Accents: The cartoon gives Bob the particular take on the Dixie accent associated with fire-and-brimstone televangelist preachers, complete with mannerisms and method of speech.
• Anticlimax Boss: Because Bob is just a little fish in a bowl, he can beaten effortlessly... if only you can get him away from his lackey, #4.
• The Brute: #4. In the games, he can't be fought, only avoided — touch him and he'll send you flying.
• Dumb Muscle: #4 is so dimwitted he can be tricked to chasing a clockwork mouse that's been tied to his tail... but is strong enough that even Jim fears getting into melee with him.
• Genius Cripple: Bob is a scientific and architectural genius, handicapped by the fact that... well, he's a tiny little fish with no limbs. He's dependent entirely on the hands of his dimwitted feline minions.
• Gone Horribly Right: In the cartoons, Bob's plans often turn up like this. Show them how to "destroy, destroy, destroy"? One of them attacks him in the same manner. Awaken the Anti-Fish? It not only refuses to obey him, but sets off to destroy the entire universe by eating the Great Worm Spirit, forcing Bob into an Enemy Mine situation with Jim if he wants to be able to conquer the universe. Mutate the entire fish population of La Planeta de Agua to have super intelligence? They realise he is a dangerous megalomaniac who must be obliterated and turn on him.
• Gratuitous Spanish: The cartoon name for Bob's world translates to "The Water Planet" or "The Planet of Water", and is always followed in show by a Mexican yell (usually written as "Arriba!").
• Harmless Villain: In the games. In the cartoons he is bumbling like all the others but can cause trouble for Jim in his schemes.
• Irony: In the cartoon, Bob's accent and mannerisms are a blatant Shout Out to a Dixie televangelist, yet he is actually a hardcore scientist and easily comparable to Professor Monkey-for-a-Head in his genius.
• Silent Antagonist: #4 and, in the games, Bob.

Game: A mad genius on retention to Queen Slug-for-a-Butt, the Professor works in a nightmarish laboratory space station, creating all manner of nightmarish creations. Working without restrictions, he indulges in all manner of insane experiments, most notably an effort to improve his intelligence by grafting a monkey to his head. This backfired terribly; he now has double the headaches, the monkey continually messes up his experiments, and he can't get the shape of bananas out of his head. At the Queen's bidding for an ultra-powerful weapon to conquer the galaxy, he created the Super Suit — and then the monkey ate the blueprints. This wouldn't have been such a bother if Psycrow hadn't gone and lost the Suit. When Jim wandered into his laboratory, the Professor eagerly tried to reclaim his handiwork, but failed.

Cartoon: A freelanced evil scientist, the Professor claims he was once a simple dairy farmer before he grafted a monkey to his head and became the most notorious of evil geniuses in the galaxy. Creator of the Super Suit, he can build another one whenever he wants with no problems — but the suit requires a Battery of the Gods to function, and he only had the one. When he tried to get another, he was turned into a human breadmaker and can, by focusing, produce warm fresh bread from inside his clothes. He notes this is actually kind of handy, as twisting the monkey's tail lets him make pumpernickel.

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• Berserk Button: In the cartoon, the Professor hates fruit carts because one killed his father.
• Domestic Abuse: In the cartoon, the relationship between the professor and the monkey isn't exactly wine and roses. The monkey has accused the professor of "making eyes at a gorilla" (to which the professor angrily retorted he's a happily grafted man), viciously beaten him with a steel rod during a psycho-therapy session, and deliberately put shrinking cream in the professor's coffee so it can watch shows the professor hates.
• From Nobody to Nightmare: In the cartoon, it's claimed he was once nothing more than a simple dairy farmer. Now, he's such a space villain he has even been featured on Lifestyles Of the Rich & Villainous, with such creations as the Sparktronic Protobluntifier, the Atomic Banana, the Pay Toilet, and, of course, the creation of the Super Suit.
• Mad Scientist
• No Plans, No Prototype, No Backup: Subverted. In the games, the professor had blueprints but the monkey ate them, while in the cartoon he can rebuild the suit whenever he wants, he just can't make them as powerful without another Battery of the Gods.
• Straw Character - Scientist: According to his blog, the creationist Doug Ten Aple created Professor Monkey-for-a-Head as a Take That to his Darwinist high school science teachers.

Game: After escaping from yet another ambush by Psycrow, Jim accidentally stumbles across a wormhole and is pulled through it, crashlanding on a strange and dangerous alien world. There, he comes across an innocuous yet friendly individual, who identifies himself as Peter Puppy and explains he has the parts Jim needs to repair his Pocket Rocket and escape this hellish place; if Jim will just escort him back home... But this is easier said then done, for Peter revealed he would transform when endangered into a mindless monster that would attack Jim. Finally reaching Peter's house, Peter was good as his word, but asked to be taken away from his nightmarish homeworld as a favor, which Jim agreed to.

Cartoon: Once an ordinary dog mocked by his fellow canines for being small and cowardly, Peter dared to break the Great Taboo of the Dogs — approaching a running vacuum cleaner — in an effort to prove himself. But vacuum cleaners are canine-specific trans-dimensional portals, and Peter was sucked through a wormhole to the Planet Heck, where Evil the Cat set a demonic spirit on Peter that transformed him into an anthromorphic dog — who happened to turn into a monster when hurt, scared or enraged. Banished back to Earth, Peter was outcast as a freak, until he stumbled into the path of Earthworm Jim. After a rather hostile first meeting, Jim warmed up to his "fellow freak of nature" and eagerly took Peter on as his sidekick.

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• Ascended Extra: From the subject of an Escort Mission in the first game to Jim's primary sidekick and righthand man.
• Barefoot Cartoon Animal: In the cartoon, at least, where he sports a shirt and shorts, but no shoes. In the games, he's completely naked.
• Butt Monkey: In the cartoon. He suffers a bit in the games too, though dishes out the abuse more often.
• Explosive Breeder: By the time of Earthworm Jim 2, Peter is the father of 600 children.
• Kid Sidekick: In the cartoon.
• Killer Rabbit: Peter looks harmless and fluffy... right up until he transforms and eats you.
• Only Sane Pup: Shares this role with Princess What's-Her-Name in the cartoon.
• Servile Snarker: In the cartoon, he can switch between being a naive follower of Jim or a neurotic commentator to his bumbling from scene to scene.
• Superpowered Evil Side: Peter's transformation is extremely strong and dangerous... and also a mindless berserker that attacks anything nearby, usually Jim. It's even caused by Demonic Possession in the cartoon.
• Vitriolic Best Buds: With Jim in the cartoon, between Jim's antics and Peter's uncontrollable transformations, there is the odd tension to say the least, but Peter certainly hates being taken from Jim's side.
  Peter: *banging his head on the table, sobbing* I WANNA BE JIM'S SIDEKICK AGAIN!!! I WANNA BE JIM'S SIDEKICK AGAIN!!!

Horrific ruler of the planet Insectica, the Queen reigns over all with a set of iron fists and she yearns to expand her dominion to the entire universe. Commissioning the creation of an ultra-powerful doomsday weapon from Professor Monkey-for-a-Head, the Queen was infuriated to learn the weapon had been lost. She seeks to destroy Earthworm Jim whenever possible as she hopes to reclaim the Super Suit, but she does not focus maniacally on that goal and will try to rule the universe in other ways.

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• Big Bad: Final boss of the game, and typically portrayed as the most important of Jim's Rogues Gallery in the cartoons.
• God Save Us from the Queen!: Somewhat subverted in the cartoons, where the Princess explains they need to reinstate Queen Slug-for-a-Butt after Psycrow and the Professor depose her because she's such a lousy evil queen that she never actually manages to conquer anything.

Game: Sweet-hearted yet ditzy sister to Queen Slug-for-a-Butt, hearing news about the Princess is what inspires Jim to travel across the universe to save her. Their first meeting, though, is less then impressive, as a cow falls on her and knocks her out cold.

Cartoon: Born hideously deformed by the standards of Insecticans, the Princess suffered countless humiliation at the hand of her malevolent sister, who refused to even allow her to have a name and banished her to the dungeons. Vowing to defeat and overthrow her sibling, the Princess trained throughout her childhood, growing up into a mighty warrior. While Earthworm Jim's insistence that they are a couple irritates her, she does consider him a good friend and appreciates his help in her efforts to try and defeat the Queen.

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• Action Girl: In the cartoon, she's the leader of a ragtag band of rebels and has basically been trying to overthrow her sister since she was a little girl.
• Brainless Beauty: In the games, according to her character profile in the manual.
• Distressed Damsel: In the games.
• Cute Monster Girl: While an Insectican like Queen Slug-for-a-Butt, she's basically a beautiful woman with long red hair, cute little wings, and a pair of antennae. In a subversion, her own species considers her hideously malformed.
• Hot Amazon: In the cartoon.
• Only Sane Woman: In the cartoons, she shares this role with Peter Puppy.
• Super Strength: In the cartoons, she boasts that as an Insectican, she has the strength of 100 men.

A strange creature that resembles a large blob of green goo with eyes, Snott is stated in the games to be a refugee from the Mucus Planet, having snuck into Jim's backpack in search of a sandwhich while Jim was recovering after defeating Major Mucus. In the cartoon, his origins are never revealed.

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• Canon Immigrant: Though Word Of God is that he was the slimy platforms that Jim battled the Queen on in the final level, for most people he only appeared in the cartoons and was then brought to the games in Earthworm Jim 2.
• Eloquent In My Native Tongue: To Jim and Peter, who can understand him, Snott can say a lot of things with only a few gibbers.
• Team Pet: His basic role in the cartoons seems to be this.
• The Unintelligible: Snott speaks a language of slurps, gurgles and burbles.

Evil the Cat's #1 lackey, the masochistic Henchrat lives to obey Evil's every command, thanking him with genuine delight for his abuse. When not on the clock, Henchrat enjoys jumping up and down on hats... while people are still wearing them.

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• Abnormal Ammo: The Henchrat's choice of weapons consist of exploding mushy cheese grenades and a gun that fires quick-setting melted cheese.
• The Brute: Henchrat serves as the muscle to Evil's brain, and is quite happy with this state of affairs.
• Canon Foreigner: Only introduced in the cartoon series.
• Hidden Depths: Henchrat enjoys macrame, watching mimes, playing piano and making balloon animals.

A twisted opposite of Jim spawned when a distorted photo-copied image of Jim was exposed to toxic waste, Evil Jim yearns to destroy his heroic counterpart.

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• Evil Twin
• Exact Opposite
• Wicked Cultured: Evil Jim can be very eloquent and mature, when he's not focused on mayhem and destruction.