Voldemort's remaining followers could be the antagonists. They might see Harry's little sister as a way to get revenge on Harry or as a potential tool for their dark purposes.
The Ministry of Magic could also play the role of antagonists if they have some sort of prejudice against Harry's family situation or if they suspect the sister has some dangerous or unregulated magical power. They could try to interfere with Harry's raising of his sister, creating all sorts of conflicts for Harry to overcome.
There could be a new dark wizard or witch emerging in the wizarding world who sees Harry's little sister as a threat because of her relation to Harry. This new antagonist could target her specifically, forcing Harry to use all his skills and resources to protect her while also trying to figure out the motives and powers of this new enemy.
Voldemort's remaining followers could be antagonists. They might target Harry's little sister thinking they can use her to get to Harry. They could try to kidnap her or use dark magic on her.
One possible antagonist could be a jealous rival among the pirate gangs. This person might envy Harry Hook's sister's skills or popularity. Additionally, a corrupt official in the Isle of the Lost who wants to keep the status quo and sees her as a troublemaker could be an antagonist. Also, if there are any ancient curses or dark spirits on the isle, they could turn against her and try to stop her from achieving her goals.
Another idea is that Harry, already being a hero, has to teach his sister all about the wizarding world. He shows her Hogwarts, but they face challenges like some students being jealous of her relation to Harry. They go on adventures in the Forbidden Forest together, and Harry imparts his wisdom and courage to her.
The Akatsuki could also be the main antagonists. They might see the power of raising the dead as a way to achieve their goals. For example, Pain could use the raised dead to spread fear and chaos, or to try and find a way to resurrect Nagato. And they would definitely be a formidable force against Naruto if they were after this power.
It could be some rogue ninjas who are after Lemon for some reason. Maybe they think she has a special power that they can use for their evil plans.
Another possibility is that there could be a new character introduced in the fanfiction, a mastermind who manipulates Harry into believing that his family is a threat. This new character could be the main antagonist.
In a 'Harry Potter and His Little Sister' fanfiction, the sister could be a new student at Hogwarts when Harry is in his later years. She struggles to fit in because of being Harry Potter's sister. Harry has to help her deal with the pressure, make friends, and also face the threat of a new dark wizard who has a vendetta against the Potter family.
It could be Draco Malfoy. He might not like the idea of Harry getting a second chance at the OWLS and tries to sabotage him. Maybe he spreads rumors about Harry not being worthy of a retake.
One plotline could be Naruto teaching his sister ninjutsu. Since Naruto has his own unique skills like the Rasengan, he could pass on some basic forms of it to his sister. Another could be Naruto protecting his sister from the village's initial distrust, just as he faced it himself. Maybe he has to stand up to the village elders to ensure his sister gets a fair chance at becoming a ninja.
Well, perhaps in the fanfiction, Harry's little sister has some unique magical ability that attracts unwanted attention. Harry then has to raise her while constantly fending off those who would use her for their own purposes. He would have to balance being a brother and a protector, making sure she grows up happy and safe in a rather dangerous magical environment. He might also introduce her to his friends at Hogwarts for support and guidance as she gets older.