thumb|300px|rightPLOT In Gotham City, Batman and Robin attempt to stop Mr. Freeze from stealing a cache of diamonds, but he escapes. The Dynamic Duo soon learn that Mr. Freeze was once a scientist named Victor Fries who became dependent on a diamond-powered subzero suit following an accident in a cryogenics lab he was using to find a cure for his wife, Nora, who was suffering from a terminal illness called MacGregor's Syndrome.
In South America, Pamela Isley is working under Dr. Jason Woodrue, a Mad Scientist who is experimenting with the Venom Drug. She witnesses Woodrue use the formula to turn a diminutive convict into a hulking monstrosity dubbed "Bane". Woodrue and Isley argue over the use of the drug and Woodrue overturns a shelf of various toxins onto her. She transforms into the beautiful and seductive Poison Ivy before killing Woodrue with a kiss from her now-poisonous lips. She finds that Wayne Enterprises funded Woodrue, so she takes Bane with her to Gotham.
Meanwhile, butler ASlfred Pennyworth's'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. Alfred is revealed to be suffering from MacGregor's Syndrome.
Poison Ivy interrupts a Wayne Enterprises press conference to propose a project that could help the environment. Bruce Wayne declines her offer, as it would kill millions of people. That night, a charity event is held by Wayne Enterprises with special guests Batman and Robin in attendance. Poison Ivy decides to seduce them. Freeze crashes the party and steals a diamond from the event. He is captured and sent to a chamber prison in Arkham asylum, but escapes with the help of Ivy and Bane.
Robin becomes smitten with Ivy and begins to rebel against Batman. Ivy imprisons Robin when he does not give in to her charms; she also subdues Batman when he arrives to save his partner. Just then, Barbara — now a costumed crimefighter calling herself Batgirl — shows up and fights Ivy, eventually defeating her.
Batman, Robin, and Batgirl decide to go after Freeze together. By the time they get to the observatory where Freeze and Bane are, Gotham is completely frozen. Robin and Batgirl confront Bane and defeat him, while Batman and Freeze begin to fight each other, with Batman winning. Batgirl and Robin unfreeze Gotham.
Batman then shows Freeze a recording of Ivy during her fight with Batgirl, in which the former brags about killing Nora. Batman then tells Freeze that his wife is not dead; she has been restored in cryogenic slumber and moved to Arkham so he can finish his research. Batman asks Freeze for the cure he has created for the first stage of MacGregor's Syndrome for Alfred; Freeze atones for his misdeeds by giving him the medicine he had developed.
Ivy is shown imprisoned in Arkham with a vengeful Freeze as her cellmate. Alfred is eventually healed. Everyone agrees to let Barbara stay at the mansion.