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- Harley Quinn (first appearance)(main story and flashback)
- Poison Ivy (single appearance)(main story and flashback)
- Ghost 1 (single appearance)(Catwoman's disguise)
- Catwoman (first full appearance)(final appearance)
- Ghost 2 (single appearance)(Batgirl's disguise)
- Batgirl (first appearance)(redeemed)
- Batman (mentioned)
- Robin (mentioned)
- Ace the Bat-Hound (mentioned)
- Joker (mentioned)
- Museum guards (single appearance)(no lines)(flashback only)
- Gotham City
- Apartment building
- Museum (flashback only)
- Bank (flashback only)
- Harley Quinn and Poison Ivy heard about the gang from the Joker, who has it out for them after they helped Batman and Robin catch him in the The New Scooby-Doo Movies episodes, The Dynamic Scooby-Doo Affair and The Caped Crusader Caper.
The title is a play on the webseries (and comic), Gotham Girls, starring the DC characters in this story.
- Instead of based on the designs of the Gotham Girls, which starred on the TV series, The New Batman Adventures, the character designs of the guest stars are based on Batman: The Animated Series, particularly Poison Ivy and Catwoman. Catwoman had a cameo appearance in Who's Scared?, where she was based on her Golden Age look from the mainstream comics.
- None known.
Inconsistencies/continuity errors and/or goofs/oddities
- Even though Harley claimed the Joker wanted vengeance on Mystery Inc., when he later shows up in Happy Harley-daze!, he doesn't even remember who they are.