A lost soul that has become extremely powerful and malevolent on the Phantom Express could be the main villain. This soul might be jealous of the living and wants to drag Cuphead, Mugman and all the other characters into the eternal darkness of the train.
There could be a hidden entity that is the true power behind the Phantom Express. It could be an ancient demon or a cursed spirit that uses the train as a means to collect souls. This entity would be the ultimate challenge for Cuphead and his friends in the fanfiction.
The conductor of the Phantom Express could be the main villain. He could be controlling the souls on the train and using them for his own dark purposes. He might be trying to spread the darkness of the Phantom Express to the rest of the Inkwell Isles.
One idea could be that Cuphead and Mugman accidentally board the Phantom Express thinking it's a regular train. Once on board, they discover the souls of the damned are the passengers. They have to find a way to get off without getting trapped themselves. Maybe they make a deal with the conductor to perform a series of impossible tasks in exchange for their freedom.
The main villain could be an older and more powerful version of Vlad. Maybe he has finally perfected his cloning techniques and created an army of half - ghost clones. He could be more devious and have a grand plan to take over both the human and ghost worlds.
A powerful warlock who wants to harness the powers of both Danny and Carrie for his own evil purposes. He could be an ancient being who has been waiting for the right time to strike when both Danny and Carrie are at their strongest.
Vlad Masters could easily be the main villain. Since he already has his own ambitions and a certain amount of power, in a royal - themed fanfiction, he could be trying to take over the ghostly royal throne. He might see Danny as a threat to his plans, especially if Danny has some newfound royal status, and thus plot against him.
It could be Sam and Tucker. Since Danny has become a villain, his former friends would likely try to stop him. They would be the main antagonists from Danny's point of view now. They would use their knowledge of Danny and their own skills to try and bring him back to the good side.
Perhaps a rogue ghost who has managed to hack into Cybertronian technology. This ghost, let's call it Techno - Ghoul, has the ability to possess and control Transformers. He starts causing chaos by making Autobots and Decepticons fight each other. Danny Phantom has to use his ghost - hunting skills and his knowledge of both the human and ghost worlds to find a way to exorcise Techno - Ghoul from the Cybertronian systems.
A corrupted version of one of their own friends could be the main antagonist. For example, a possessed Boris from Bendy's world or a mind - controlled Mugman from Cuphead's world. This would add an emotional layer as they have to fight someone they care about.
Perhaps a mad scientist from Danny's world who has somehow gotten access to Ultraman technology and is using it to create chaos. He could be creating mutant creatures that are a threat to both the Ghost Zone and Ultraman's world, and Danny and Ultraman have to stop him before it's too late.
Another possible antagonist could be the new opera managers. They could be trying to use Christine's disappearance to their advantage, perhaps by spreading rumors that she has run away with a lover to create a scandal and boost ticket sales. They might also try to stop Raoul from searching for her, seeing it as bad for business.
A possible main villain could be a dark wizard from Star's world who has somehow gained the ability to control ghosts. He could use this power to try and take over both Danny's world and Star's, seeing the combination of magic and the ghost zone as a means to ultimate power.