Four years after the defeat of the Enchantress, the Joker breaks up with Harley Quinn, throwing her out on the Gotham City streets. She is taken in by Doc, the owner of a Taiwanese restaurant, and recovers from her relationship by cutting her hair, adopting a spotted hyena, and taking up roller derby.

Harley gets drunk at a nightclub owned by ruthless crime lord Roman Sionis, and cripples his driver after he insults her. She meets burlesque singer Dinah Lance, who later rescues an intoxicated Harley from an attempted abduction. Impressed by Dinah’s fighting skills, Roman appoints her as his new driver. The next night, Harley gets drunk again, and overhearing some of her drinking buddies debating over whether she has actually broken up with the Joker, blows up the Ace Chemicals plant to publicly announce their breakup. When everyone finds out she is not under the Joker’s protection anymore, Harley starts getting attacked by every criminal that holds a grudge against her.

Meanwhile, GCPD Detective Renee Montoya investigates a series of mob killings carried out by a crossbow-wielding vigilante. She tries to recruit Dinah as an informant, but she rejects the offer, as the GCPD wasn’t there to prevent her mother’s death. Roman sends Dinah and his sadistic right-hand-man Victor Zsasz to retrieve a diamond embedded with the account numbers to the fortune of the Bertinelli crime family, who were massacred years ago. Young pickpocket Cassandra “Cass” Cain steals the diamond from Zsasz and swallows it after she is arrested.

Harley, fleeing from Montoya and several others, is captured by Roman’s men. Roman sends Harley to recover the diamond for him, under the threat of death, and also places a bounty on Cass. Breaking into the GCPD with a grenade launcher filled with paint and sparkle rounds, Harley frees Cass and the pair escape. While hiding out at Harley’s apartment, they bond. Doc is approached for information by the “crossbow killer”, who is revealed to be Helena Bertinelli. Having survived her family’s massacre and becoming trained as an assassin, Helena has been targeting each of the gangsters responsible for her family’s murders, preferring the moniker “Huntress”. Harley’s place is later bombed by criminals looking for Cass, and Doc admits that he sold Harley out. Harley calls Roman and offers to turn her over in exchange for his protection, agreeing to meet at an abandoned amusement park. Dinah notifies Montoya of the rendezvous, but her betrayal is noticed by Zsasz, who informs Roman. A devastated Roman dons his ritualistic mask from which he gets his nickname, “Black Mask”.

At the park, Montoya confronts Harley, but is kicked out of a window. Zsasz arrives and tranquilizes Harley before holding Dinah at gunpoint, but he is killed by Helena, who reveals he was the last of her family’s killers. Montoya returns and reveals that Roman was the true mastermind behind the Bertinellis’s massacre. He had them killed trying to get the diamond. A stand-off ensues until they realize Roman has arrived with a small army of masked criminals. Using Harley’s old gear, the makeshift team of five women withstand and repel the attack. During the battle, Cass is captured by Roman, while Dinah reveals her metahuman ability of supersonic-level screaming, defeating several of the mobsters. Harley gives chase on roller skates, and with assistance from Helena, the pair pursue Roman. At a nearby pier, Roman prepares to kill Cass, who pulls the ring from a grenade that she took from Harley’s weapons chest. Harley throws him off the pier just before the grenade detonates, tearing him apart and killing him.

In the aftermath of destroying Roman’s criminal empire, Montoya quits the GCPD. Using the money from the accounts hidden inside the diamond, Helena joins with Dinah and Montoya in establishing a team of vigilantes called the Birds of Prey. Harley and Cass escape, selling the diamond itself to a pawn shop and starting their own business.

In the post-credits scene during the credits rolling, Harley narrates at the audience, wondering why they are still there, and just when she is about to say something about Batman, she gets cut off.