There could be an ancient evil entity that is awakened by the union of Naruto and the Vampire Queen's powers. This entity wants to plunge both the ninja world and the vampire world into chaos and will stop at nothing to destroy Naruto and the Queen, using all kinds of dark magic and illusions to try and defeat them.
Maybe a dark ninja clan that has a grudge against Naruto and also wants to control the Vampire Queen for her powers. They would try to set traps for both Naruto and the Queen, making it difficult for them to trust each other and work together against the real threat.
It could be the other demons who oppose the Queen of Demons' newfound alliance with Naruto. They might see Naruto as a threat to their way of life or as a tool that the Queen of Demons is misusing. So, they would try to break their bond and destroy Naruto.
It could be a powerful dark wizard or sorcerer who wants to control both Naruto's world and the Spider Queen's domain. This antagonist might be after the unique powers that Naruto and the Spider Queen possess, like Naruto's chakra and the Spider Queen's web - based magic. He could try to manipulate them against each other to achieve his goals.
It could be a powerful demon who wants to control the Succubus Queen and use her powers for evil. Since Naruto is with the Queen, he becomes a target too.
It could be Kaguya Otsutsuki. Since she has powerful abilities and a thirst for power, she would see Kefla's power as a threat and Naruto's influence as something to be eliminated. She might try to manipulate events to turn the ninja world against Kefla and Naruto.
It could be someone who wants to merge their two worlds in a chaotic way. Maybe an evil scientist from Leela's world who has discovered the power of chakra and wants to use it for his own dark purposes. He could try to capture Naruto to study his chakra and use it to take over both universes.
The main antagonist could be an evil merman who wants to take over Ariel's kingdom and is also jealous of her friendship with Naruto. He might use his dark water - based magic to try and stop them.
An ancient evil force that was sealed away by Hashirama long ago could be released in Naruto's time. This entity, having a grudge against Hashirama for imprisoning it, would see Naruto as an obstacle too since he's allied with Hashirama. It could have powers that are completely unknown to the modern ninja world.
It could be Salem herself if the fanfiction focuses on Naruto trying to stop her evil plans. Since Salem has a long history of causing chaos and destruction, Naruto would see her as a force to be reckoned with and try to oppose her.
It could be a rogue ninja from an old clan who has a vendetta against both Konoha and Kumogakure. This ninja has been gathering power in secret and plans to destroy both villages to avenge his clan's past defeat at the hands of the combined forces of the two villages.
It could be a character like Orochimaru who somehow gets access to Gotham's technology and becomes even more powerful. He could be creating new types of jutsu using the advanced tech there and causing chaos in both worlds.