Harley Quinn who has finally broken things off once and for all with the Joker and attempts to make it on her own as the criminal Queenpin of Gotham City. The series features Harley Quinn, Poison Ivy and a whole cast of heroes and villains, old and new, from the DC Universe.
- Kaley Cuoco as Harleen Quinzel / Harley Quinn: The main protagonist who is the runt of the chain gang in Arkham Asylum at the very beginning of the series of the same name. However, as the series progresses, she recently escapes custody with her cellmate, Ivy, and sets off on a most epic adventure into becoming criminal Queenpin of Gotham City.
- Lake Bell as Pamela Isley / Poison Ivy: Sarcastic and incredibly smart, Ivy is Harley’s best friend and now roommate. Very much a loner (she prefers plants over people), Ivy is sometimes the voice of reason but always a true friend to Harley. Ivy soon becomes her reluctant partner-in-crime.
- Alan Tudyk as:
- The Joker: Equal parts homicidal and charismatic, he’s been Harley’s longtime awful boyfriend, but when Harley breaks up with him, she vows to take his cherished number-one spot on the Most Wanted list. The Joker thinks of Harley as his creation and, as she grows in power, does everything he can to prevent it from happening;
- Basil Karlo / Clayface: A large, humanoid mass of clay, Clayface was a struggling, trained actor until a terrible pottery accident turned him into the shapeshifting villain he is now. His aspirations for stardom and his ability to transform his appearance to look like anyone should help him land some roles but he’s such a terrible actor that the only work he can get is with Harley’s crew, pretending to be whoever they need him to be help with a heist. He still holds out hope that he’ll land one of the many auditions he fruitlessly goes on.
- Julian Day / Calendar Man
- Condiment King
- Tom Kenny as Clayface's hand
- Jason Alexander as Sy Borgman: Once upon a time, Sy used to be a suave, super spy. Now in his 90s, he’s a wheelchair user with most of his body replaced by creaky 50s era bionic parts. Crotchety, difficult and longs to become relevant again. He’s also Harley and Ivy’s landlord, and insists they take him on their heists so can get back into the action – or else he won’t repair the plumbing.
- J. B. Smoove as Frank the Plant
- Ron Funches as King Shark: His lower half is human, but his upper half is the body of a great white shark. Even though he looks incredibly fierce, he’s a gentle soul who just wants to be normal. That is, until he smells blood. And then he goes into a frenzy and literally eats everyone around him leaving death and carnage everywhere. Then feels bad about it.
- Tony Hale as Dr. Psycho: An angry misogynist dwarf with telekinesis. He was a major villain until the “incident” ten years ago when in the midst of losing a battle against Wonder Woman he became frustrated and called her the C-word. In front of hundreds of camera phones. Even other villains were disgusted. Disgraced, the only one who’ll have him is Harley. A woman. He can’t stand it, but he’ll do anything to get back into the villain community.
- Diedrich Bader as Bruce Wayne / Batman: A fully deputized special agent to the Gotham Police Department who turned Harley in and brought her to the Arkham Asylum for a life sentence behind bars prior to this series. His alter-ego, Bruce Wayne, is a director to a community service program which enforces Arkham's inmates under hard labor until they are allowed to return to society.
- Wanda Sykes as Tsaritsa / Queen of Fables
- Natalie Morales as Lois Lane
- Jim Rash as Edward Nigma / The Riddler
- Giancarlo Esposito as Lex Luthor
- Christopher Meloni as Commissioner James Gordon
- Rahul Kohli as Jonathan Crane / Scarecrow
- Sanna Lathan as Selina Kyle / Catwoman
- Vanessa Marshall as Princess Diana / Wonder Woman
- Sean Schemmel as Dr. Jeremiah Arkham: Harley's loudmouth warden of the Arkham Asylum who acts as a drill instructor. Although, he runs this prison in extreme inhuman measures, he forces hard repeating labor on inmates, to torture them both physically and mentally, until they lose their criminal ways and reenter society. In fact, he aims to reform people, not to offer pure torment.
- Jacob Tremblay as Damian Wayne / Robin: The boy wonder and Batman's sidekick who helped turn Harley Quinn in prior to the series. At the beginning of the first episode, Harley was ordered by Batman to stand on a platform scale with height meter so that he can measure her weight and height for a correct sized prison uniform.
- James Adomian as Bane
- Matt Oberg as
- Tisha Campbell-Martin as Tawny Young and MONICA
- Phil LaMarr as Black Manta
- Briana Cuoco as Barbara Gordon / Batgirl
- Andy Dale as Harvey Dent / Two-Face
- Wayne Knight as Oswald Cobblepot / The Penguin
- Sean Giamborne as Joshua Cobblepot