Antagonists
Lawrence Lactavius Limburger (voiced by W. Morgan Sheppard) – The main villain of the series and archenemy of the Biker Mice. Lawrence Limburger is a bloated alien from Plutark and has an odd fascination for Earth's criminal society of the 1930s. He is the head of Limburger Industries, the biggest industrial company in Chicago. Wearing a purple business suit and a rubber mask in order to look human at most times, he attempts to mine it and other parts of Earth for various resources, including random earth, dirt, rock, snow, metal, and oil, which he plans to send back to his own dying planet. He is highly resourceful and cunning, but always fails due to the Biker Mice's interventions. During the War of Liberation on Mars, Limburger was a low-ranked and bullied officer, taunted by both his superior Stilton and Karbunkle. Limburger managed to attain his present rank by using Stilton's absence during the Biker Mice's attack on the Plutarkian base to attain his present rank. Limburger reappears in the 2006 sequel series, initially as a bootlicker for the Pit Boss after the Catatonians came into view in a flashback and later as a co-conspirator alongside the Catatonians.
Dr. Benjamin Boris Zachary Karbunkle (voiced by Susan Silo) – A thin, sly mad scientist of an indeterminate humanoid species. Karbunkle used to work for Limburger's superior Dominic T. Stilton until Limburger bribed him to cross over. Karbunkle's main task is to think of machines and robots to either battle the Biker Mice, gather resources from Earth, or look up the villain of the week with his dimensional transporter. During the War of Liberation on Mars, he gave Modo his bionic arm and eye, Stoker his bionic tail, and Throttle his malfunctioning bionic eyes as part of his experiments to create a bionic army after they were captured by Plutarkian forces. He intended to use one of his inventions, the Mind Bender Beam, to brainwash them into being loyal to the Plutarkians; he was only able to brainwash Stoker before the Biker Mice escaped, and Stoker was later freed from Plutarkian control, while the Beam was destroyed. Karbunkle is a sycophant, often seen admiring Limburger and praising him; however, Karbunkle's top priority is Karbunkle. He is often seen torturing Fred the Mutant, cares little about his co-worker Greasepit, and has traded Plutarkian employers many times.
Greasepit (voiced by Brad Garrett) bungling sidekick usually in charge of whatever project Lawrence Limburger is running at the time. Greasepit rides a motorized tricycle into battle against the Biker Mice at the head of his goons and wears a pair of gardening trousers like a mechanic's. As his name suggests, Greasepit constantly oozes oil, and he is consistently clumsy, often falling over or dropping critical pieces of Limburger's schemes.
Fred the Mutant (voiced by Rob Paulsen) – A masochistic mutant working for Limburger. He thrills at the thought of receiving pain, and that is his one purpose as well, being the subject of many of Karbunkle's testings as well as physical abuse by Limburger. Fred has three eyes, a bushy tail, duck-like feet, and a tentacle in place of a right arm.
Napoleon Brie (voiced by Luke Perry) – Limburger's unhinged chief Plutarkian rival from Detroit who is far more successful than Limburger in his efforts. He dominates Detroit. Despite this, Brie's attempts at taking out the Biker Mice turn out just as futile as Limburger's. Brie's efforts are not helped by his undermining Limburger at the same time. Small of stature and the owner of a large variety of facial masks, although he only ever wears the one with a crazy eye and speaks with an accent very similar to Elmer Fudd. Brie reappears in the 2006 series, where he and Limburger had a brief truce with the Catatonians.
Number One (voiced by Mark Hamill in the first appearance, Jeff Bennett in later appearances) - A gun for hire with a big red beard and sunglasses who works for Napoleon Brie.
Plutarkians – The Plutarkians are fish-like aliens that either plan to obtain Earth's resources or conquer Earth. Each of them is named after a cheese. Besides Limburger and Brie, the Plutarkians consist of:
Lord Camembert (voiced by Jeff Bennett) – The Plutarkians' supreme ruler and Limburger's higher-up. In some other cases, he will appear with the entire Plutarkian council with Limburger in a panic over what task is current. He often appears on the vidcom or in person to Limburger and berates him for his failure.
Mudfish Murdock (voiced by Jeff Bennett)—A Plutarkian trucker who was hired by Limburger to kill the Biker Mice while he attempted to drain Lake Michigan and deliver it to Plutark.
Provolone (voiced by Jeff Bennett)—A Plutarkian lawyer.
Dominic T. Stilton (voiced by Malcolm McDowell) - A Plutarkian who was Limburger and Karbunkle's superior on Mars prior to the events of the series. Stilton hates Limburger for his scheming and repeated attempts to usurp him. Unlike other Plutarkians, Stilton is a neat freak who constantly carries around handkerchiefs to wipe dirt and grime off of himself and anything he touches.
Gerald Guyere (voiced by Jim Cummings) - A Plutarkian operating in New Orleans.
Jack Monteray (voiced by Jeff Bennett) - A Plutarkian operating in Las Vegas. He is often nicknamed "One-Eyed Jack".
Gutama Gouda (voiced by Jim Cummings) - A Plutarkian operating in Los Angeles.
Romana Parmesana (voiced by Tori Spelling in normal form, Jennie Garth as Angel Revson) - A Plutarkian operating in Cleveland. She used her disguise of Angel Revson to manipulate Jack McCyber in her plot to usurp Lawrence Limburger and Napoleon Brie.
Pit Boss (voiced by Stu Rosen) – The Pit Boss is the burly ruler of the Pits outside of town. The Pit Boss has an electric whip that he uses to keep his slaves in line.
Pit Crew – The henchmen of Pit Boss who will come up and rob various places, often taken hostages as well.
Road Ravens - A gang that was once used by Limburger to steal gasoline from different trucks.
Jimmy Mac (voiced by Jerry Houser) - The leader of the Road Ravens.
Stalkers (voiced by Robert Ito and Brad Garrett respectively) - A group of alien bounty hunters.
Mace (voiced by Jeff Bennett) – Mace is a nomadic rat who appears to be disguised as a mouse and into intervening with the mice and the Plutarkian war in secret and sometimes coming into conflict with Vinnie, but he escaped by grabbing Harley and teleporting to the Nomad Rat base.
X-Terminator (voiced by Dorian Harewood impersonating Arnold Schwarzenegger) – A robotic bounty hunter who rides a customized combat motorcycle.
Lectromag (voiced by Mark Hamill) – An electromagnetic supervillain who the Biker Mice often have a hard time defeating.
Tunnel Rat (voiced by Rob Paulsen) – A rat-like villain who is an expert at driving any digging vehicle.
The Loogie Brothers (voiced by Neil Ross and Jess Harnell) – Hacka and Honka Loogie are known as the "Scum of the Universe." They were summoned by Limburger to stink up Chicago, summoned to collaborate with Evil Eye Weevil in a motocross plot, and later summoned to take advantage of Chicago's garbage crisis.
Evil Eye Weevil (voiced by Jess Harnell impersonating Elvis Presley) – An egotistical Martian villain and former stunt performer who can induce hostility in anyone. Limburger summoned him to break the Biker Mice apart. He claims to be Elvis Presley's alien brother and dresses like and has similar mannerisms to Presley. Evil Eye Weevil later collaborated with the Loogie Brothers in Limburger's motocross plot.
Pukes of Hazard (voiced by Jeff Bennett and Rob Paulsen) – The aides of Evil Eye Weevil.
Corroder Cody (voiced by Charlie Adler) – An eyepatch-wearing villain of indeterminate human-shaped species who was hired by Limburger to purloin the oil refineries.
Munsterella and Gorgonzola (voiced by Elinor Donahue and Eddie Barth) – A towering reptilian humanoid duo.
Weathermeister (voiced by Russi Taylor) – A female villain of indeterminate human-shaped species and old friend of Karbunkle. She can control the weather by placing her weather stickers on a map.
Pulverizer (voiced by Jeff Bennett) – A cyborg. He assisted Limburger into stealing a military weapon called the Annihilator.
Stone Cutter (voiced by CCH Pounder) – A female jackhammer-wielding supervillain. Limburger enlisted her to help steal Mount Rushmore.
Jet Blaster (voiced by Jeff Bennett) – A robotic villain with no legs who can fly unusual devices. He was hired by Limburger to cut Chicago from Earth so that he can send it to Plutark.
Catscan (voiced by Jeff Bennett) – A cat-like villain with psychic powers that was used by Limburger in a plot to get the Last Chance garage condemned.
Slobber the Mutt (voiced by W. Morgan Sheppard) - The leader of the hyena-like Sand Raiders on Mars.
Billie Monnie (voiced by Susan Silo) – A bird-like bounty hunter. She was originally after Limburger for an unknown client until Limburger paid her to target the Biker Mice.
Mechanic – A mute cyborg who can control mechanical devices.
Cycletaurs - Creatures created by Karbunkle that are the result of him combining the DNA of the Biker Mice with the molecular structure of three motorcycles. In the episode "Diet of Worms", a Cycletaur was paired up with Gutama Gouda.
Icebreaker (voiced by David Warner) – A supervillain who has powers over cold and heat.