There could be a new group of villains. Maybe they are a secret organization that has been watching Ash for a long time. They fear that his secret could disrupt their plans for world domination in the Pokemon world. So, they constantly try to stop him from uncovering his own secret.
One possible main antagonist could be a powerful and arrogant trainer who wants to prove that he is better than Ash and Nora. He might try to steal their Pokemon or sabotage their battles.
Perhaps a group of bandits who steal Pokemon and sell them on the black market. They see Ash and Serena as threats because they are trying to stop their illegal activities. These bandits could be led by a really tough and mean - looking guy who has no mercy when it comes to getting what he wants. They would constantly be trying to sabotage Ash and Serena's attempts to protect the Pokemon and the people in the medieval setting.
Often, Team Rocket can still be a main antagonist. They might come up with new and devious plans to steal Ash's Pikachu or Serena's Pokemon. Their comical yet persistent attempts to cause trouble add a lot of action and humor to the fanfiction.
Perhaps a powerful and mysterious trainer. This trainer is jealous of Ash's connection with his Pokemon and his ability to conduct these experiments. He wants to prove that he is the better trainer. So he tries to disrupt Ash's experiments by challenging him to battles at inconvenient times. He also spreads false rumors about Ash's experiments being dangerous to other Pokemon, which causes some local Pokemon communities to be hostile towards Ash.
It could be Team Rocket. They are always causing trouble for Ash and his Pokemon in the Pokemon series, so it's likely they would be the antagonists in this fanfiction too.
One type of antagonist could be a corrupt Pokemon League official. In the fanfic, this official might bribe Ash to do something unethical, like throwing a battle against Iris. When Iris finds out, she'll feel betrayed. Another likely antagonist is a Pokemon hunter. If the hunter captures a Pokemon that Ash and Iris both care about, and Ash, in an attempt to rescue it, makes a deal with the hunter that seems to go against Iris's values, she'll feel betrayed. And sometimes, it could be a self - centered Pokemon trainer who wants to be the only star. They might set up Ash and Iris to fight each other and make it look like one has betrayed the other.
Maybe Ash has some hidden powers or a special origin that makes him secretly more than just a regular trainer. For example, he could be part Pokemon himself, which gives him an edge in understanding and communicating with them on a deeper level.
Team Rocket is very likely. They've always been after Ash and his Pokémon. Using mind control on Ash would be a new way for them to get what they want. They could make Ash give them Pikachu or other strong Pokémon. Another one could be Giovanni. As the leader of Team Rocket, he has the resources and the motive to use mind control on Ash to further his own evil plans in the Pokémon world.
His friends like Misty and Brock would probably visit him.
A group of rogue scientists who are experimenting on Pokemon in unethical ways could be new antagonists. They might be creating powerful and dangerous hybrid Pokemon.