Another possibility is that the main antagonist is a group of poachers who are trying to capture the spiders for illegal magical trading. They don't care about the balance of the wizarding world or Harry's new - found role. Their actions are endangering the spiders, and Harry has to stop them. They might be using advanced dark - magic - based traps and weapons that Harry has to overcome in order to protect the spiders and the forest.
It could be a powerful dark wizard who wants to control the spiders for his own evil purposes. He might see Harry as a threat to his plans since Harry is the 'King of the Spiders'. This wizard could be using dark spells to manipulate the spiders and turn them against Harry and the wizarding world.
Maybe the main antagonist is a giant, mutated spider that has broken away from the normal spider hierarchy. It sees Harry as an intruder in its territory and is determined to get rid of him. This spider could have unique and terrifying abilities, like being able to spit a venom that can erase memories or paralyze wizards. It would constantly be hunting Harry and his allies throughout the fanfiction.