Antagonists
- Bane (voiced by Joaquim de Almeida in "Traction", Ron Perlman in "Team Penguin", Clancy Brown in "The Batman/Superman Story") is a South American mercenary who derives superhuman strength from the Venom steroid.
- Black Mask (voiced by James Remar) is a meticulous and ruthless crime lord. He has no fingerprints or distinguishing features, making him difficult to identify.
- Blaze (voiced by Rachael MacFarlane) is a disgraced nuclear physicist and ally of Firefly.
- Catwoman (voiced by Gina Gershon) is a professional cat burglar and activist.
- Clayface (voiced by Wallace Langham in "Clayfaces" and Lex Lang in "The Batman/Superman Story") is a failed actor who steals a sample of Joker Putty and gains superpowers in a bid to gain attention.
- Cluemaster (voiced by Glenn Shadix as an adult and Kath Soucie as a child) is a former game show contestant who seeks revenge on those he believes rigged the show and cost him his title as champion.
- Captain Slash (voiced by Diedrich Bader) is a villain from an in-universe video game.
- Count Dracula (voiced by Peter Stormare) is a deceased vampire who the Penguin accidentally resurrects using his blood.
- Count Vertigo (voiced by Greg Ellis) is a former employee of Queen Industries who uses an eyepiece to induce vertigo.
- D.A.V.E. (voiced by Jeff Bennett) is a robot created by Hugo Strange and programmed with the personalities and knowledge of several of Batman's adversaries.
- Everywhere Man (voiced by Brandon Routh) is a physicist and friend of Bruce Wayne. He creates a machine that can duplicate matter and uses it to clone himself, only for one of the clones to become evil and take his place.
- Firefly (voiced by Jason Marsden) is an arsonist who wields a bug-like suit. Late in the series, he steals a phosphorus isotope that transforms him into a metahuman with innate fire abilities.
- Gearhead (voiced by Will Friedle) is a criminal racer who can hack into vehicles and possesses retractable claws. The Batman Strikes! reveals him to be a former underground racing celebrity who was injured in an accident and rendered quadriplegic before an unknown doctor gave him cybernetic implants.
- Mercy Graves (voiced by Gwendoline Yeo) is Lex Luthor's bodyguard.
- Francis Grey (voiced by Dave Foley) is a clockmaker and thief who stole a pocket watch and was arrested for a larger crime that he accidentally caused. After being imprisoned for years, Grey develops the ability to rewind time and battles Batman before altering the timeline so that he was never arrested.
- Harley Quinn (voiced by Hynden Walch) is a television psychiatrist who the Joker recruits to become his sidekick.
- The Joining is an extraterrestrial technology-based collective entity intent on absorbing Earth.
- The Joker (voiced by Kevin Michael Richardson) is a villain bent on causing chaos and making people laugh. He wields various gadgets, but is equally skilled in combat, using his feet as dexterously as his hands to increase his mobility.
- Hideo Katsu (voiced by Keone Young) is the head of a Yakuza crime syndicate.
- The Kabuki Twins are Penguin's silent Asian henchwomen.
- Killer Croc (voiced by Ron Perlman) is an anthropomorphic crocodile and crime boss. He rarely works on his own, tending to work with other villains.
- Killer Moth (voiced by Jeff Bennett) is a low-tier servant of the Penguin. He later transforms into a monstrous anthropomorphic moth after being exposed to radiation.
- Lex Luthor (voiced by Clancy Brown) is an amoral businessman and enemy of Superman.
- Man-Bat (voiced by Peter MacNicol) is a Wayne Enterprises scientist who transforms himself into a monstrous anthropomorphic bat in an attempt to be feared like Batman. Later in the series, he reforms and becomes Batman's ally.
- Marty (voiced by Patton Oswalt) is a technician and the Joker's assistant.
- Metallo (voiced by Lex Lang) is a Kryptonite-powered cyborg and enemy of Superman.
- Mirror Master (voiced by John Larroquette) is a mirror-themed villain and enemy of the Flash.
- Mister Freeze (voiced by Clancy Brown) is a professional thief who previously gained ice-based abilities after being locked in a cryonic freezer. In a possible future depicted in the episode "Artifacts", Freeze's body deteriorates and he begins using mechanical spider-like legs.
- Number One (voiced by Diedrich Bader in "The Breakout" and John Mariano in "What Goes Up...") is the name of several henchmen who serve Black Mask.
- The Penguin (voiced by Tom Kenny) is an aristocratic member of the Cobblepot family who turns to crime to rebuild his family's reputation and wealth.
- Poison Ivy (voiced by Piera Coppola) is an environmental activist and classmate of Barbara Gordon who gains the ability to manipulate plants after being exposed to a plant-based mutagen.
- Prank (voiced by Michael Reisz) is a classmate of Barbara Gordon who the Joker temporarily recruits as his sidekick.
- Punch and Judy are the Joker's silent henchwomen.
- Rag Doll (voiced by Jeff Bennett) is a thief and contortionist who can bend his body to great lengths.
- Rhino and Mugsy (both voiced by John DiMaggio) are the Ventriloquist's henchmen.
- The Riddlemen are the Riddler's silent henchmen.
- The Riddler (voiced by Robert Englund) is a former university scientist who seeks revenge for his prototype memory-enhancing device being sabotaged.
- Rumor (voiced by Ron Perlman) is a vigilante who seeks to kill all of Gotham's villains. He was created in place of Hush, who could not be used in the series. [6]
- Scorn (voiced by Daryl Sabara) is the younger brother and sidekick of William Mallory / Wrath.
- Shadow Thief (voiced by Diedrich Bader) is a thief who can transform into shadows and an enemy of Hawkman.
- Sinestro (voiced by Miguel Ferrer) is a Yellow Lantern with fear-based abilities and an enemy of the Green Lantern Corps.
- Smoke (voiced by Amanda Anka) is the Mirror Master's henchwoman.
- Solomon Grundy (voiced by Kevin Grevioux) is a zombie and the subject of an urban legend stating that he was created to get revenge on Gotham's founders for polluting the environment. Ethan Bennett impersonates Grundy in the episode "Grundy's Night", while the real Grundy appears in The Batman Strikes!.
- Spellbinder (voiced by Michael Massee) is a mystic with a third eye that allows him to create illusions and control the minds of others.
- Hugo Strange (voiced initially by Frank Gorshin and subsequently by Richard Green following Gorshin's death) is the head psychiatrist of Arkham Asylum. He is more interested in discovering how the criminal mind works than helping to cure the inmates.
- Temblor (voiced by Jim Cummings) is a mercenary who wields armored gauntlets.
- The Terrible Trio are university students who use variants of Kirk Langstrom's formula to transform into anthropomorphic animals and terrorize their classmates. They consist of David / Fox (voiced by David Faustino), Justin / Shark (voiced by Googy Gress), and Amber / Vulture (voiced by Grey DeLisle).
- Rupert Thorne (voiced by Victor Brandt) is an aging crime lord who Batman defeats at the beginning of the series.
- Cosmo Krank / Toymaker (voiced by Patton Oswalt) is a toymaker who produces dangerous toys and seeks revenge on Bruce Wayne for shutting down his company. Krank also appears in Gotham , portrayed by Chris Perfetti, while shops bearing his name appear in the Batman: Arkham series. [7]
- Toyman (voiced by Richard Green) is an evil toymaker and enemy of Superman.
- Ventriloquist (voiced by Dan Castellaneta) is a former ventriloquist who snapped and became a criminal after being booed off stage.
- Wrath (voiced by Christopher Gorham) is the older son of a pair of jewel thieves who were arrested and jailed around the same time Bruce Wayne's parents were killed. He and his younger brother Andy / Scorn become criminals to help other criminals, believing that they have a right to profit as much as heroes and civilians.
- Maxie Zeus (voiced by Phil LaMarr) is an eccentric billionaire and former mayoral candidate who is obsessed with Greek mythology and culture.
- Tony Zucco (voiced by Mark Hamill) is a crime boss and former circus performer who killed the Flying Graysons.
