Maybe a mechanical tyrant in a steampunk - inspired setting. This tyrant has created an army of mechanical beasts and is trying to rule the city. Pyro, as a girl with fire powers, is the only one who can melt the metal parts of the beasts and stop the tyrant. The tyrant sees her as a threat to his rule and is constantly trying to eliminate her.
It could be an envious rival. Maybe there's another girl who is jealous of Pyro's unique abilities or charm. She constantly tries to undermine Pyro and create obstacles in her path.
It could be an original character created by the fanfic writer. Maybe a young girl who discovers her pyro powers after a strange event.
Maybe a malevolent scientist who has a grudge against female heroes. He believes that only men should have such powers and sets out to take the Omnitrix from Ben and use it for his own evil purposes. He underestimates Ben because she is a girl but soon realizes his mistake.
It could also be a new villain that is specifically interested in Sonic because she is now a girl. Maybe a villain who has some sort of vendetta against female heroes or wants to use Sonic's new form for some evil experiment. However, Dr. Eggman is so iconic in the Sonic series that he would be a strong contender for the main antagonist role even in this fanfic scenario.
It could be an evil wizard who was the one who actually caused Gumball to turn into a girl in the first place. The wizard has some ulterior motives, like using Gumball's new form to gain power or to cause chaos in the town. He tries to prevent Gumball from finding a way to turn back and keeps creating obstacles for Gumball and her friends.
It could be a villain who is obsessed with creating chaos through strange transformations. This villain might have targeted Deku specifically because of his status as a rising hero. They want to see how Deku will cope with being turned into a girl and use it to undermine his confidence and hero image.
It could be a character who somehow discovers Izuku's secret and tries to use it against her. Maybe a rival at school who is jealous of Izuku's progress and sees this as a way to get her expelled or discredited in the hero community.
Another possibility is that an alien race that Ben has previously encountered and thought was friendly, now turns against him/her because of the change in gender. They might have some strange beliefs or customs that make them view Ben's new form as a threat or an abomination, and thus they become the main antagonists in the fanfic.
Ganon could still be the main antagonist. Even though Link has turned into a girl, Ganon's goal of taking over Hyrule remains the same. He might underestimate Link in her new form, which could give Link an advantage. For example, Ganon might not expect the girl - Link to be as strong or resourceful as the male Link, but she proves him wrong.
Perhaps a jealous classmate who has always been in Izuku's shadow. Now that Izuku is a girl, this classmate sees an opportunity to undermine her. They spread rumors about how Izuku's transformation is some sort of cheat or that she's not the same reliable person she was as a boy. This classmate's actions and attempts to sabotage Izuku would make them the main antagonist.