Early in his career, Spidey fought a trio of criminal mercenaries called the Enforcers rather often, Fancy Dan, Montana, and the original Ox, the last little more than a Dumb Muscle thug named Raymond Bloch. Eventually, Ox left the other two to join Mr. Fear's Fellowship of Fear, where he was killed — or so it seemed — from a fall during a battle with Daredevil. It turned out, however, that Raymond had a twin brother named Ronald who was also a Dumb Muscle thug, but whom Raymond had never mentioned. Nonetheless, Ronald joined up with the other two members of the Enforcers to take his brother's place. (And stayed with them when Raymond turned up alive years later, working for The Kingpin.)
The opening storyline of Spider-Man’s Tangled Web introduced another of Peter Parker's classmates who was there during the spider-bite incident. Unfortunately this was a super-smart sadistic bully. The nutbar figured things out and ate the now dead power-granting spider, becoming a body-hopping crazed killer. It got worse from there.
Original Sin introduced us to Cindy Moon, who would go on to become Silk. She was introduced as another person who was there at the experiment that gave Peter his powers and was even bitten by the same spider before it died. She was picked up by Ezekiel Sims in an attempt to protect her from Morlun and never bothered to tell anyone connected to him.
Carlie Cooper was supposedly good friends with Gwen Stacy, and Gwen's dad Captain Stacy supposedly worked with Carlie's dad as well.
Subverted with Clayton Cole, who first appeared in The Amazing Spider-Man (Dan Slott), a 2014-debuting teenager who was also "introduced" as having been around during Peter's high school/early Spider-Man days. This mechanically gifted but otherwise chronically shy and introverted, home-schooled teen was one of Spidey's first and "biggest"note from Clayton's perspective, not so much Spidey's fans from his wrestling days, and even helped (in his mind) spread the transitioning Superhero's popularity. Deciding he wants to be a hero, too (in part to be close to his idol, but also for fame soaking-up purposes), he creates his own costumed identity, complete with homemade sound-based powers, dubbing himself "Clash". Alas, his first meeting in-costume with Spidey didn't go so well, ending with Spidey declaring/treating the bumbling newbie as just another villain. The popularity his Clash persona ends up receiving going straight to his head coupled with Spidey spurning him causes Clayton to snap, culminating into the final "confrontation" at school, with Spidey unmasking the newly-dubbed "Creepy Clayton". This Trope is subverted in Clayton's case because, despite the trappings, no one actually remembers Clayton; for Peter, he was nothing more than an early career blip that largely resolved itself. Clayton also has more of a happier ending later in his life then many similar others, and despite his and Spidey's history, doesn't really become more than a minor supporting character in Peter's life.Details After the unmasking, Clayton ends up in juvie. He's on parole 13 years later, and due to his circumstances, has to hire himself out as a masked scientist goon to get any work. Having largely matured and mellowed out since his short-lived, yet life-defining Clash days, he turns on his employer and helps Spider-Man foil her plans. In turn, Spider-Man, who's also matured vastly from his teenage years, directs the now-bearded Clayton (whom he does eventually recognize) toward Parker Industries, where an equally-matured Peter welcomes him with open arms. Thus, Clayton is now a loyal, if somewhat minor (compared to a few of his fellow employees), scientist working for Parker Industries, albeit while still on parole (so he cannot be seen in any costume, even if he helps Peter/Spidey out in a crisis situation, a la the Ghost sabotage).
In Mark Waid and James Robinson's Amazing Spider-Man: Family Business, it is revealed that Spider-Man supposedly has a long-lost younger sister named Teresa. Supposedly, because it is unclear whether or not Teresa is really his sister since Mentallo was responsible for putting a perception filter effect on her.
End of the Spider-Verse: Literally with Spider-Boy. He’s apparently from Earth-616 and allied with Peter and Miles, but was somehow severed from the Great Web for so long, no one remembered him.
The Janice Lincoln version of the Beetle is an interesting example. She was around as a minor villain for a little while, but wasn't revealed to be the daughter of Tombstone (a fairly prominent Spider-Man villain) until she started appearing in The Superior Foes of Spider-Man. The fact that we'd never seen her before was subtly lampshaded, as it turns out that her dad purposefully steered her away from the world of costumed supervillainy so that she could make even more money as an attorney.
The Shuri solo series introduces a kid named Augustine Torres, a Child Prodigy who attended the same school as Miles Morales and is supposedly one of his close friends.
Venom: Eddie Brock was arguably this upon his initial introduction as Venom; his now-infamous origin story was connected with the earlier Sin-Eater arc, but Eddie was never in that arc when it was running, and his backstory was a Retcon. His first ever appearence was after he gained the symbiote-suit. Peter knew who he was when Eddie finally unmasked, but the audience had to be told via-flashback to events that apparently happened off-page during the Sin-Eater arc.
Issue 26 of Venom (Donny Cates) sees a character calling himself "Virus" be talked about as Venom already met him, who says that Eddie ruined his life. First off, this is in part due to the fact there was a FCBD special that was meant to be released before issue 26 that's have featured Venom's first meeting with Virus. Secondly, it's subverted with the reveal that he's really Mac Gargan, the Scorpion/third Venom.
Venom (2021): In Venom #2, Eddie's voicemail sends Dylan to California in search of an old friend of his from his days as a reporter, Archer Lyle... who has never previously appeared or been mentioned.
