Two years after defeating Two-Face and the Riddler, Batman and his new partner, Robin, come into conflict in the form of a new foe, Mr. Freeze, who has left a string of diamond robberies in his wake. During a confrontation in the natural history museum, Mr. Freeze steals a bigger diamond and flees, freezing Robin and leaving Batman unable to pursue him. Later, Batman and Robin learn that Mr. Freeze was originally Dr. Victor Fries, a doctor working to develop a cure for MacGregor’s syndrome to heal his terminally ill wife. After a lab accident, however, Fries was rendered unable to live at normal temperatures and forced to wear a cryogenic suit powered by diamonds in order to survive.
At a Wayne Enterprises lab in Brazil, botanist Dr. Pamela Isley is working under the deranged Dr. Jason Woodrue, experimenting with a drug named Venom. She witnesses Woodrue use the formula to turn the violent, but diminutive, convicted serial murderer Antonio Diego into a hulking monstrosity, who he dubs “Bane”. When Isley threatens to expose Woodrue’s experiments, he attempts to kill her by overturning a shelf of various toxins. Despite Woodrue’s efforts, Isley is resurrected, transforming into the beautiful and seductive Poison Ivy before exacting revenge; she kills Woodrue with her poisonous kiss, and sets fire to the lab, leaving it to burn down while she escapes with Bane. She finds that Wayne Enterprises funded Woodrue, though they cut Woodrue’s funding when he intended to weaponize the Venom drug, thus she appropriates Bane as a muscle-bound thug, taking him with her to Gotham City. Meanwhile, Alfred Pennyworth’s niece, Barbara Wilson, makes a surprise visit and is invited by Bruce Wayne to stay at Wayne Manor until she goes back to school.
Wayne Enterprises presents a new telescope for Gotham Observatory at a press conference interrupted by Isley. She proposes a project that could help the environment, but Bruce declines her offer, which would kill millions of people. Batman says that this project is in conflict with the main goals of the Wayne Corporation. That night, a charity event is held by Wayne Enterprises with special guests, Batman and Robin, and she decides to use her abilities to seduce them. Mr. Freeze crashes the party and steals a diamond from the event. Although he is captured by Batman and detained in Arkham Asylum, he eventually escapes with the help of Poison Ivy, who killed two security guards with her kiss in the process. Meanwhile, Dick discovers that Barbara has participated in drag races to raise money for Alfred, who is dying of MacGregor’s syndrome.
Batman and Robin begin to have crime fighting relationship problems because of Ivy’s seductive ability with Robin, but Bruce eventually convinces Dick to trust him. Poison Ivy is then able to contact Robin once more; she kisses him but fails to kill him due to Robin wearing rubber lips. Robin says that the rubber lips are immune to her charms. Meanwhile, Barbara discovers the Batcave, where an AI version of Alfred reveals he has made Barbara her own suit. Barbara dons the suit and becomes Batgirl. Ivy captures Robin, but he gets rescued by Batman, and Batgirl arrives and subdues Ivy to get eaten by her throne plant, before revealing her identity to the pair.
Batman, Robin and Batgirl decide to go after Mr. Freeze together. By the time they get to the observatory where Mr. Freeze and Bane are, Gotham is completely frozen. Batgirl and Robin are attacked by Bane, but they eventually defeat him by kicking apart his venom tubes, stopping the flow of venom to his body. Bane collapses before reverting to his original form and is left helpless on the ground.
Meanwhile Batman and Mr. Freeze begin to fight each other, with Batman defeating Mr. Freeze. Batgirl and Robin manage to thaw the city, and Batman shows Freeze a recording of Poison Ivy during her fight with Batgirl, who had informed the latter that she killed Mr. Freeze’s wife. However, Batman informs Mr. Freeze that she is still alive, in cryogenic slumber before being moved to Arkham Asylum, waiting for Mr. Freeze to finish his research. Batman proceeds to ask Mr. Freeze for the cure Mr. Freeze has created for the first stage of MacGregor’s Syndrome to administer to Alfred, and Mr. Freeze atones for his misunderstanding by giving him the medicine he had developed.
Mr. Freeze is then detained in Arkham Asylum. Poison Ivy is also imprisoned in Arkham Asylum with a vengeful Mr. Freeze as her cellmate (due to his own cell is in the process of being modified for his laboratory needs) and he plans to make Poison Ivy’s life a living hell while staying for the attempted murder of his wife. After Alfred is cured, everyone agrees to let Barbara stay at Wayne Manor and fight crime with them.