The Punisher seemingly has one purpose in life: he kills bad guys, knowing that most of the time the system doesn't work. After Frank Castle's wife and children died at the hands of mobsters, he set out to make sure villains died before they could hurt anyone else. This included some very powerful people, both heroes and villains.
Most of Punisher's big-league kills have come in the pages of alternate timeline stories. These include What If...? issues, as well as one-shot stories like Punisher Kills the Marvel Universe. However, there are other notable fatalities that came in both the mainstream Marvel Universe and the Ultimate Universe, showing that the Punisher is as strong as any superpowered being when he has his heart set on a target.
Barracuda
In Punisher: Barracuda, Frank Castle met his match in the powerful killer Barracuda. This was from the Punisher MAX series, which takes place outside the main Marvel continuity. The series was one of the best Punisher series of all time, with Frank trying to bring down corporate criminals.
The final fight ended up a ship with Punisher finally outsmarting the corporate bad guy and killing him. Baraccuda even helps Punisher in the end when the bad guys betrayed him. However, Punisher doesn't forgive the villain, who was put out of his misery by a sea of hungry sharks. Barracuda's death is Castle at his most unforgiving. Frank doesn't care if Barracuda showed signs of remorse; all that matters is justice being served by any means necessary.
Himself
Punisher is his own worst enemy. He has a thirst for vengeance and wouldn't stop until he had killed every villain that needed to be taken down. In the alternate world series Punisher Kills the Marvel Universe, he set out to kill all the superheroes, blaming them for his family's death when an Avengers battle had collateral damage.
His last kill was one that shocked him. Punisher killed Daredevil, not knowing it was Matt Murdock, the attorney who represented him and someone that Frank trusted. After this, Frank realized what he had become and knew there was one more guilty person who needed to die — himself.
Wilson Fisk
Frank Castle in The Punisher MAX storyline was ruthless and made his mainstream counterpart look almost heroic in comparison. In the regular Marvel Universe, Wilson Fisk, the Kingpin of Crime, is almost untouchable and extremely powerful. In Punisher MAX, he was just another target.
The Punisher MAX #22 saw Frank and Fisk in a major fight to the finish. It was a bloody fight and Castle ripped through Kingpin's minions, taking a few bullets along the way. Finally, the two confronted each other with no distractions. Castle bit off Fisk's tongue, stabbed him with a claw hammer, and then put several bullets into the back of his head. Kingpin's grisly death illustrated just how low Castle had sunk as the thin line between antihero and villain that had always defined him was obliterated.
Spider-Man
Punisher first appeared in one of the best Spider-Man comics in the 1970s, setting out to kill the Web-Slinger when the villain Jackal convinced Frank that Spider-Man had murdered Norman Osborn in cold blood. However, Punisher figured things out and let Spidey live. That wasn't true in every timeline.
In Marvel Vs. The Punisher, Spider-Man was patient zero during an outbreak that turned superheroes into cannibalistic monsters. It turned out that in the case of Spider-Man, his actions were mostly to save Mary Jane from Kingpin, which he did with Punisher's help. However, after this, Punisher killed Spider-Man anyway, saying he was a monster and had to die.
Captain America
In the Marvel Universe vs. The Punisher storyline, Frank Castle was trying to stop the superheroes who caught the disease that turned them into cannibalistic monsters. The strongest of these villains is Hulk and Punisher teams with Captain America, Deadpool, and Wolverine to stop him.
Deadpool and Wolverine set out to find Hulk and leaves Punisher and Captain America to fight an infected mob. However, Cap realizes he is infected and is changing. He asks Punisher to end it before he can turn and Punisher does so, telling Cap that he was always his hero. This murder was probably the most tragic as Frank was killing the very symbol of truth and justice he had believed in even when he was a vigilante.
The Thing
The Thing was one of the most powerful superheroes affected by the virus from Marvel Universe vs. The Punisher. The Fantastic Four had stopped Spider-Man after his initial infection, but Thing ended up infected and then joined Hulk to go on a rampage, trying to kill scientists looking for a cure.
Punisher has very little problem killing regular people when he sets his mind to it. However, Ben Grimm is made of solid rock and is almost invulnerable. Punisher finally found a way to stop the Thing by shooting an armor-piercing missile into the Baxter Building and killed Ben that way. Ben's death, while gruesome, showcases one of the vigilante's underrated qualities: his strategic mind. He found a way to breach Grimm's almost impenetrable skin that had perplexed more powerful foes like Dr. Doom.
Wolverine
Wolverine is someone that a person can kill, but he will always be back. The Punisher knows this, so he realizes that he can kill Wolverine if he has to and not feel bad because Logan will always be back. That doesn't mean it is easy because Wolverine is one of the most powerful and persistent mutants alive.
In Punisher #16-17 by Garth Ennis and Darick Robertson, Punisher and Wolverine went to battle and Punisher held nothing back. He started out by shooting Wolverine in the face with a shotgun. Before Logan could regenerate, Punisher had a steamroller barrel over the mutant, squashing and him leaving him dead until Wolverine eventually regenerated after Punisher had finished his current mission.
The X-Men
One of the essential What If...? issues from its initial run asked what would happen if Wolverine was turned into a vampire. With the vampire powers added to his already immense healing factor and rage-induced feral nature, he was almost unstoppable. Wolverine then turned most of the X-Men into his own vampire army.
After Doctor Strange died, his spirit went to the astral plane and possessed Punisher to keep fighting. Punisher destroyed most of the X-Men vampires with holy water guns and silver bullets, single-handily destroying one of the most popular Marvel teams of all time.
The Hulk
In Punisher Kills the Marvel Universe, Hulk was one of the people that Punisher killed on his way through the universe. However, this wasn't the only time he did so and it wasn't even the most impressive. In that series, Punisher shot Bruce Banner, which should have immediately brought out the Hulk.
In Secret Wars: Battleworld #1, Punisher had a quicker way to wipe out Hulk. In this series, Punisher had the mystical power of Doctor Strange. When Hulk showed up, Punisher turned him back into Banner and then turned him to dust without a second thought. While Hulk is Earth's strongest Avenger, it makes fans wonder if Doctor Strange has been holding back all this time.
The Marvel Universe
When Punisher killed Hulk, that was a big deal. However, he also went through and killed every other member of the Marvel Universe before he was finished. In a battle at a New York park, the Avengers and X-Men were there and Frank's family was collateral damage.
He immediately killed Cyclops and then went to jail. When he got out, he targeted everyone else. He killed Spider-Man and Venom, gunned down Captain America, and even murdered Daredevil, not realizing it was his friend Matt Murdock. The biggest kill came when the X-Men was summoned to the moon, only to realize it was a trap and Punisher nuked the moon, killing all the heroes that were marooned there. This over-the-top massacre was typical of alternate time storylines like this one that saw various characters freely slaughter heroes and villains without much consequence