Bo-Bo is a child of seeming contradictions- he's male, but he dresses in what seems to be a dress (it's actually a Pierrot's blouse, or so he insists. He wears white-face clown makeup), it's repeatedly alluded to that he's Asian (and that the name "Bo-Bo" is just his own surname, repeated- "that's not how you pronounce it! I'm being clever!" (I can see him alluding to the actual pronunciation with him saying "bye-bye", which would be how it would be pronounced if he did repeat his name)), but he speaks in a heavy Bostonian accent, peppered with gratuitous Italian. Personality-wise, he's cheerful, distractible, and very affectionate with his friends- when he does get otherwise emotional, though, he's very dramatic about it. I can see him having generous homoerotic subtext with other male characters, part for comedy, and part because I actually do see him as being queer? He can jump high, and maybe fly for short periods of time, and has a bullet-hell adjacent attack style, complete with abstraction- quickly shooting out abstract projectiles, such as cute stars, at the player.
Nightshade is a sarcastic chlorokinetic- her outfit is a biker jacket over a somewhat ripped petal dress, floral net tights with rips in them, sunglasses, and a headband. She has mommy issues, and has the most intimate knowledge of normal crime, the two facts being related. She's the prickliest character in the game, probably, and a joke should be made based around that, with roses having thorns. She summons vines and such in her boss fight, using them for both barriers and some of her attacks. During her attacks, as well as shortly after, the barriers are down, leaving her vulnerable, though you can also just destroy the vines, which also delays her vine-based attacks (which are her stronger ones).
Jess dresses like a cowboy, complete with a bandana, and acts as the leader of the villains- he says he's the direct descendant of Jesse James, but such claims are unverified. As the leader, he's somewhat self-sacrificing for his friends, always putting them first, and acts as a sort of mediator for the team, in addition to having the final say on most decisions, and being the idea guy. He fights using an old-timey revolver, and acts as the straight man and voice of reason to the villains- he's the oldest of the villains, though still thirteen at most, and definitely not older than Gnowoman, though he could be her age rather than younger.
Kitten can turn invisible and plays heavily into the cat theme- she sneaks up on people, even among her fellow bad guys, and has a bit of a rivalry with Nightshade, who she feels needs to lighten up. She's a bit of a prankster, as well as a kleptomaniac, and has a crush on Jess. Able to turn invisible in her boss fight, against most heroes her sprite vanishes (her hit box is still there and moves like normal), but MOTHY can still see her outline.
Gimmick's gender is ambiguous- androgynous and flamboyant, they often change outfits between scenes, and fights with a mix of attacks from all of the villains- during their boss fight, they're surrounded by floating cards, and when they use an attack from one of the other villains, they flip the card with the emblem of one of the other villains, to show which attack they're going to use; Princess can prevent that attack by destroying the card. Gimmick can also attack using the cards as projectiles, throwing them. Gimmick will sometimes vibe with Bo-Bo and Kitten, but they have a trait those two lack, in that they are An Asshole. They still care about the others, but it's buried pretty deep down, which could be an element of Innocent Malice the other villains lack; they're the youngest villain, only a little older than Princess.