An old enemy of the Omnitrix's creator could be a great main antagonist. Maybe this enemy was locked away for centuries and now has escaped and blames the Omnitrix for his imprisonment. He would be extremely powerful and have knowledge of the Omnitrix's weaknesses that Ben doesn't even know yet.
Well, Ben is obviously one of the main characters as the story is centered around him and the Omnitrix. Gwen might also be a main character as she often plays an important role in Ben's adventures, providing support or using her own magical abilities to help out. Grandpa Max could be another main character, offering his wisdom and experience to guide Ben through various situations.
It could be Vilgax. He might see Ben 10's new form as a weakness and try to take advantage of it.
Hex could also be a main antagonist. He might be jealous of the Lucky Girl's natural powers and try to corrupt her or use her powers for his own evil magic. He could set up traps and illusions to try and capture her, and also try to turn Ben against her so he can deal with them separately.
The main antagonist could be an alien who has a vendetta against Ben specifically. This alien might be able to sense Ben's true identity even when he's dressed as a girl. He could try to expose Ben and foil his mission or just cause general chaos while Ben is in his disguised state.
One idea could be that the Omnitrix malfunctions and gives Ben new, unexpected alien forms with powers he has to quickly learn to control. For example, an alien that can manipulate time in small bursts, which would create really cool scenarios where he has to fix things that went wrong in the past few seconds. Another idea is that a new villain is specifically targeting the Omnitrix and Ben has to protect it while also trying to figure out the villain's motives. Maybe this villain has a device that can nullify the Omnitrix's powers temporarily.
Maybe a malevolent scientist who has a grudge against female heroes. He believes that only men should have such powers and sets out to take the Omnitrix from Ben and use it for his own evil purposes. He underestimates Ben because she is a girl but soon realizes his mistake.
Well, obviously Ben Tennyson is a very popular character. He is the main character who wields the Omnitrix after all. His different alien forms like Four Arms or Heatblast are also quite prominent in the fanfiction world.
One possibility is that the story could explore how Gwen's personality changes when she has the Omnitrix. She might become more confident or more responsible. Also, there could be new villains who are attracted to the Omnitrix now that Gwen has it. She would then have to face these threats and protect the Omnitrix from falling into the wrong hands.
It could be an ancient evil that has been awakened by the chaos of the two universes colliding. This entity is powerful and has the ability to manipulate the very fabric of the Gears of War and Ben Carmine's world. It wants to destroy both worlds and reshape them in its own dark image, and Ben Carmine and the Gears have to figure out a way to stop it before it's too late.
Another possibility is that an alien race that Ben has previously encountered and thought was friendly, now turns against him/her because of the change in gender. They might have some strange beliefs or customs that make them view Ben's new form as a threat or an abomination, and thus they become the main antagonists in the fanfic.