Maybe there's a new evil organization in the fanfiction. They could be after Aggron because of its strength. They might want to use Aggron's power for their own evil purposes, like taking over the Pokemon world.
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.
It could be that Aggron gets into some trouble, like being captured by an evil team. Ash then has to come up with a plan to rescue it. He might use his other Pokemon and his wits to outsmart the bad guys. Along the way, we could see how much Ash cares for Aggron and how far he'll go to save it.
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.
There could be a rogue legendary Pokemon that sees Ash's transformation as a threat to the natural order. This legendary Pokemon, with its own set of motives, could be trying to prevent Ash from becoming a fully - fledged legendary - like being. It might set up various obstacles and battles for Ash, testing his will and strength at every turn.
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.
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.
Obviously, Ash is one of the main characters. He's the protagonist who has a passion for pokemon and is always eager to become a pokemon master. And the legendary pokemon he encounters or befriends would also be important characters. For instance, if it's Lugia, Lugia would play a major role in the story as it's a very powerful and mysterious pokemon.
Obviously, the Legendary Pokemon would be a main character. It could be a Pokemon like Mewtwo, who is often depicted as having a complex personality. It might start off as a mysterious and powerful entity that Ash has to befriend or battle against.