Perhaps a jealous rival. This person might be a male who is threatened by Ed's success or popularity. When they discover Ed is a girl, they could use this secret to try and drive her out of the group or community she's in. They might spread rumors or create problems for her.
Maybe there's a dark wizard who has a vendetta against Natsu's family. When he finds out that Natsu is secretly a girl, he sees it as an opportunity to further his evil plans. He could try to kidnap her or use her as a bargaining chip against her family or friends. He might think that because she's a girl, she'll be easier to manipulate or capture compared to if she was thought to be male.
Alphonse could be a major supporting character. Since he is Ed's brother, he would be in a unique position to either help her keep the secret or accidentally almost expose it. Winry could also be important. She might be someone Ed could confide in or struggle to keep the secret from, especially if their relationship takes on new dimensions due to Ed's secret gender.
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.
Perhaps a rival adventurer who discovers Bilbo's secret and tries to use it against her. This antagonist could be jealous of Bilbo's place in the group and sees her secret as a way to undermine her and take her position. He might spread rumors or try to create situations where Bilbo's secret is likely to be revealed.
Perhaps an old enemy of the Sannin resurfaces. This antagonist could see Naruto's attempts to follow in the Sannin's footsteps as a threat. They might target Naruto to get revenge on the Sannin or to prevent a new powerful force from emerging in the ninja world. For example, if there were a group that was once defeated by the combined efforts of the Sannin, they could try to stop Naruto before he becomes as strong as the Sannin were together.
It could be someone who wants to expose Naruto's secret. Maybe a rogue ninja who wants to use this information to create chaos in the village by making the villagers doubt Naruto's intentions.
Perhaps a rogue Frontier Brain who has his own ideas about how the Frontier should be run. He might see Ash as a threat to his plans, especially if Ash is secretly learning so much about the Frontier Brains. This antagonist could try to sabotage Ash's journey and his secret training.
It could be a rogue shinobi from Naruto's past who has been biding his time. This shinobi might have developed a new jutsu specifically designed to target Naruto and make him die slowly. He's doing it secretly to cause chaos in the Hidden Leaf when their hero suddenly passes away.
The Cullens could potentially be the main antagonists. Since Bella is secretly a Volturi fan, the Cullens' views and actions that oppose the Volturi might be seen as obstacles in Bella's new - found admiration. For example, if the Cullens try to keep Bella away from Volturi - related things, they would be acting against her interests.
A powerful sorcerer who wants to enslave all the insects for his own dark magic could also be the main villain. Mosqito Girl would have to use her wits and powers to stop him.