It could be Aku. Since he is the main villain in Samurai Jack's world, his power and evil nature would make him a great antagonist. He could see Harry's magic as a threat and try to eliminate him. And if he teams up with some of the dark forces from the Harry Potter universe like Voldemort or the Death Eaters, it would be even more challenging for Harry and Jack.
Voldemort could also be the main antagonist. Imagine if he found a way to enter the world of Samurai Jack and started to spread his brand of dark magic there. He might try to enslave the people in Jack's world and use their resources to become even more powerful. Harry would have to convince Jack that Voldemort is a great threat and together they would have to fight against Voldemort's army of Death Eaters and dark creatures.
Another possibility is a new character created specifically for this fanfiction. This character could be a powerful sorcerer who has mastered both the dark arts from the Harry Potter world and the dark magic in Samurai Jack's world. He could be the one pulling the strings behind the scenes, making both Voldemort and Aku do his bidding. Harry and Jack would first have to figure out this new villain's identity and then find a way to defeat him.
It could be a dark fairy who wants to use the power of both the wizarding world and Pixie Hollow for her own evil purposes. She might try to steal the magic from Hogwarts and the pixie dust from Pixie Hollow to become all - powerful.
Maybe the main antagonist could be Ra's al Ghul. He's always interested in power, and if he got his hands on some magic from the Harry Potter universe, he could become an even greater threat. He could try to use the magic to bring about his vision of a 'perfect' world, which would be at odds with both Harry's and Nightwing's ideas of justice. So they'd have to join forces to stop him from achieving his goals.
It could be a descendant of a rival family to the Peverells. This person might be jealous of the power and secrets that the Peverell family held and wants to stop Harry from uncovering them. They could try to manipulate events to their advantage and create obstacles for Harry.
There could be a group of dark wizards who are working together to bring chaos to the wizarding world. They target Amelia Bones because of her position in the Ministry and Harry Potter because of his fame. This group, with their evil intentions and powerful dark magic, would serve as the main antagonists in the fanfiction.
Perhaps a rogue Overwatch agent who has turned against the organization. When Harry arrives, this agent sees an opportunity. He believes that by using Harry's magic, he can take over Overwatch and rule the world. So he tries to manipulate Harry and turn him against Tracer and the rest of the good - hearted Overwatch members.
One possible plot could be that Harry Potter is somehow transported to the world of Samurai Jack. He teams up with Jack to fight Aku. Harry uses his magic, like the Patronus charm to fend off Aku's dark minions, while Jack uses his sword skills. Their combined powers and different fighting styles make them a formidable duo against Aku's tyranny.
It could still be Voldemort. Even though the focus is on Harry and Nagini, Voldemort is Nagini's master and the main source of evil in the Harry Potter world. He would likely try to stop Harry from getting too close to Nagini or from finding out any secrets Nagini might hold.
It could be a dark wizard who is jealous of the bond between Harry and Lily. This wizard might try to break their connection and use Lily's power for his own evil purposes. Maybe he wants to control Lily's magic, which is known to be strong, to achieve domination over the wizarding world.
It could be Voldemort who somehow follows Harry into the realm of the Morrigan. His dark magic could disrupt the balance there and make him a common enemy for both Harry and the Morrigan.
There could be a group of anti - magic fanatics in the fanfiction. They see Byako as an abomination because of his strange powers that don't conform to the normal rules of wizarding magic. They also view the Hogwarts students' acceptance of Byako as a sign of the corruption of magic. So they set out to eliminate Byako and anyone who stands in their way.