Four years after the defeat of the Enchantress,^[a] 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 (whom she names after Bruce Wayne), and taking up roller derby.

Harley gets drunk at a nightclub owned by Roman Sionis, a ruthless crime lord, and cripples his driver after he insults her. She meets burlesque singer Dinah Lance, who later rescues an intoxicated Harley from an attempted abduction. Sionis is impressed by Dinah's fighting skills and appoints her as his new driver. The next night, Harley blows up the Ace Chemicals plant as a way to publicly announce her breakup with Joker. Meanwhile, GCPD Detective Renee Montoya investigates a series of mob killings carried out by a crossbow-wielding vigilante. Finding Harley's necklace at the scene of the Ace Chemicals explosion, Montoya notes that Harley is in danger without the Joker's protection. She tries to recruit Dinah as an informant, but Dinah rejects the offer since she is upset with the GCPD for not being there to prevent her mother's death.

Sionis 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 other people she had wronged, is captured by Sionis' men. Zsasz informs Sionis that Cassandra has the diamond, and Dinah warns Montoya. Sionis kidnaps and makes Harley recover the diamond for him, under the threat of death, and also places a bounty on Cassandra. Breaking into the GCPD with a variety of firework-inspired non-lethal grenade launcher rounds, Harley frees Cassandra and the pair escape.

After escaping, Harley and Cassandra bond while hiding out at the former's apartment. 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 apartment is later bombed by criminals looking for Cassandra, and Doc sorrowfully reveals that he sold Harley out. Harley calls Sionis and offers to turn Cassandra 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 Sionis. A devastated Sionis dons his ritualistic mask from which he gets his nickname, "Black Mask".

At the park, Montoya confronts Harley, but Harley knocks her out of a window. Zsasz arrives and tranquilizes Harley before holding Dinah at gunpoint, but he is killed by Helena, who reveals Zsasz was the last of her family's killers. Montoya returns and reveals that Sionis was the true mastermind behind Helena's family's massacre and he had them killed trying to get the diamond. A stand-off ensues until they realize Sionis has arrived with a small army of masked criminals. Using Harley's old gear, the makeshift team successfully withstand and repel their attack.