The Green Goblin could be a great main villain. He could be working on a plan that not only threatens Spider - Man's world but also the areas the Defenders protect. His maniacal nature and flying abilities would pose a great challenge to both Spider - Man's acrobatics and the Defenders' combat skills.
In a spiderman defenders crossover fanfiction, the plot might involve a magical threat. Maybe an ancient evil has been awakened in the sewers of New York. Spider - Man stumbles upon it first while swinging around. He tries to fight it but quickly realizes he needs help. The Defenders, who are more in tune with the street - level, darker side of the city, are also aware of the danger. They all come together. Spider - Man can use his scientific knowledge to help figure out the origin of the threat, while the Defenders use their combat skills and street smarts to take on the minions of the ancient evil.
Maybe a biologist gone bad. He has been studying the worm and discovers that its cells can be used to create a powerful mutagen that can turn ordinary people into super - strong minions. He plans to create an army and take over the city. Spiderman has to stop him from using the worm in this way. And the worm, being an innocent creature, becomes a key piece in this battle between good and evil.
Doctor Octopus would also work well. He could use his mechanical arms to manipulate the game environment and create deadly traps for Spiderman, all while trying to steal some valuable in - game technology.
It could be Doctor Octopus. Imagine he somehow gets his hands on some Sky High technology that enhances his mechanical arms and gives him new powers. He then decides to take on both Spider - Man and the Sky High students because he sees them as threats to his new - found power and his plans for world domination.
Lex Luthor could be a great villain. He could be after some technology in Spiderman's world that he wants to use to enhance his power in his own battles against Superman. At the same time, he might see Wonder Woman as a threat to his plans of world domination. He could use his intelligence to create complex traps and weapons. His corporate power would also allow him to manipulate situations in both the worlds of Spiderman and Wonder Woman.
A powerful demon lord from the underworld could be the main villain. This demon lord could be seeking to expand his territory into the Naruto world, using his dark magic and hordes of minions.
In a crossover with the Transformers, Megatron could be the main villain. He might be interested in stealing Inspector Gadget's advanced human - made gadgets to use against the Autobots. Inspector Gadget would have to use his wits and his gadgets, along with the help of the Autobots, to stop Megatron's evil scheme.
The Reverse - Flash could be a great main villain. He could travel to Gotham to cause chaos and draw out the Flash. His time - manipulation abilities would pose a huge threat not only to the Flash but also to Batman's carefully planned strategies in Gotham. Batman would have to adapt to dealing with a villain who can move at super - speed and mess with time.
Maybe it could be a time - traveling criminal. This villain has the ability to jump between different timelines and has noticed that by disrupting the key events in both the Arrow and Avengers universes, he can create chaos and then rule over the resulting mess. He would constantly be one step ahead of the heroes as he could manipulate time to his advantage. He could also use his knowledge of future events to set traps for the Avengers and the Arrow team.
A new meta - human villain created specifically for the crossover could be interesting. This villain could have the power to control shadows and use them to move quickly, mimicking the Flash's speed in a way. In Gotham, he could use the city's many dark corners and alleys to his advantage. He could also be a master of disguise, fooling both the Flash and Batman. His motives could be to take over both Gotham and Central City and create a new world order where meta - humans rule over normal humans.