Adam: Legend of the Blue Marvel: Neutronium, also known by some as Iso-8, an unstable element from outside conventional reality, is one of the few things that can actually hurt Blue Marvel (Adam Bernard Brashear).
The Avengers: Doctor Druid's magic has a special vulnerability to iron, as did the powers of his ancestors.
Cosmic Ghost Rider: Brute force doesn't work that well on him, as Hulk and Thor found out, but Frank is vulnerable to some supernatural attacks, especially if they affect the dead.
Deadpool: Wade Wilson / Deadpool katanas are often made of carbonation, the Soviet counterpart to adamantium, mainly because carbonation's radioactivity makes it dangerous to those with a Healing Factor.
Heroes Reborn (2021): Besides magic, Hyperion (Marcus Milton)'s vulnerable to Vibranium - a substance native to his planet, which is extremely toxic to him.
The Incredible Hulk: In Immortal Hulk, solar radiation tremendously weakens the Devil Hulk/Immortal Hulk incarnation and even turns him back to Banner. Iron Man and Shadow Base exploit this.
Ms. Marvel: In Ms. Marvel (2014), electric shocks reduce the elasticity of Kamala's cells, nullifying her many of her shape-shifting powers for a few moments. The Inventor predicts and exploits this, and Kaboom's attacks trigger it at least once (though oddly, not every time).
Spider-Man: There is a certain gas that can remove Spider-Man's Spider-Sense temporarily. It is often used by the goblin-based villains (Green Goblin, Hobgoblin etc.) or by Mysterio. Venom and Carnage are completely immune to the sense altogether, since their powers come from the alien suit Spider-Man wore. Removing his spider sense or being immune to it is always shown to be disastrous for Spider-Man. For instance, his Aunt May is immune to his spider-sense due to being a trusted family member. Trying to get to his bedroom, he snuck into her house one night, still dressed as Spider-Man. She managed to sneak up on him and ended up whacking him over the head with a vase, KO-ing him. One of the plot points in Spider-Island was Peter temporarily losing his Spider-Sense and realizing just how much he depended on it. He spent some time training with Shang-Chi to become less reliant on it.
Since he's part spider, he's more sensitive to pesticides than a regular person.
Cyclops' optic blasts can be entirely contained by even a thin sheet of ruby-quartz (and, uh, his eyelids apparently) This is used to his advantage, as he can't normally control his optic blasts at all, and so wears glasses with lenses made of the material. Occasionally a villain will strap on some ruby-quartz armor, or lock a quartz helmet on to Cyclops's head, but usually they prefer to go the route of knocking off the glasses. His brother Alex is also immune to his powers, as is he to Alex's. This is explicitly a result of the shared Required Secondary Powers that stop them from blowing themselves up.
Colossus, one of the most powerful of the X-Men, can turn his body into living steel, and cannot be hurt by many things when he is in his metal form. The anti-metal vibranium, however, can hurt him, and when faced with a vibranium weapon, he instinctively reverts to his human form.
Wolverine, X-23, and some other fast healers have trouble healing when having "strange (or foreign) metals (or substances)" on their composition (such as Wolverine's adamantium skeleton, or being struck by carbonium) and thus not heal as fast (read: instantly).
Doctor Strange: Time Master powers really screw with Dormammu. Unfortunately for him, Strange brings the best kind (the Eye of Agamotto / Time Stone) for their confrontation.
Iron Man: Although the Iron Man armors can withstand things ranging from small arms fire to Thor's lightning, they are susceptible to heat intense enough to melt through them. This is why any of the Extremis fighters can almost fight on even ground with Tony Stark / Iron Man.
