She was present when Shredder and Ra's al Ghul passed through the facility, and mischievously beckoned Shredder to her cell so she could check her makeup in the reflection of his metal mask. When the Joker was released from his own high-security cell, he immediately freed Quinzel and gave her the responsibility of placing the mutagen in the Arkham sprinkler system. She complied, but not until he called her "doctor" instead of "nurse."
She mutated into a hyena, and greeted Batman and the Ninja Turtles upon their arrival. She then withdrew to a large room where the Joker and Two-Face were waiting for Batman, where she comforted her lover when he was disappointed by Batman's lack of reaction to his new form.
Quinzel's attempt to fight Batman was interrupted by the arrival of Mr. Freeze, and she gleefully watched as Batman was forcibly mutated into a crazed giant bat. She was unsure whether to be pleased when he threw Two-Face through a window, pointing out that he was on their side, but upon being told that it didn't matter by the Joker, she began laughing.
She then intervened when she saw Leonardo attempting to dose Batman with the retromutagen, crushing his hand with her mallet when he reached for the syringe. She was then attacked by Robin, who punched her repeatedly in the face but was unable to get the retromutagen from her hand. Robin bit her finger, kicked her face and managed to get his hands on the syringe, which was eventually given to Batman. Before he demutated, Batman ran into Quinzel and the Joker, smashing them into a balcony.
Though it is not shown, she was presumably demutated by the police department along with the other criminals, once the retromutagen was proven safe.
- Harley Quinn's voice actress, Tara Strong, also voices Poison Ivy in the same film.
- Tara also voiced Harley Quinn in various DC media, including her Arkhamverse counterpart from Batman: Arkham City onwards.