So out of general interest I put together a list of what I think is all the villains that have appeared in a Spider-Man game in the last 20 years or so. The games I included were Spider-Man 1 for PS1 & N64, Spider-Man 2: Enter Electro, Spider-Man: The Movie, Spider-Man 2: The Game, Ultimate Spider-Man, Spider-Man 3, Spider-Man: Web of Shadows, Spider-Man Shattered Dimensions, Spider-Man: Edge of Time, and both of the Amazing Spider-Man games. I did not include Friend or Foe as I could not find a list of all villain characters.
I did not play all of these games (but a lot of them) so the villain-lists comes from Wikipedia. The characters are listed by name and how many times they appeared as a fight able boss, plus appereance in game without being in a fight. The exception is Black Cat since I do not know in what games you fight her and in what games she just makes an appearance.
Furthermore various incarnations of different characters are counted as the same, for example Noir Goblin in Shattered Dimensions is listed as Green Goblin/Norman Osborn. The female version of Doc. Ock in Shattered Dimensions is also listed under Doc. Ock so the list is fairly arbitrary.
Total number of games counted: 11
- Rhino: 5+1
- Venom/Anti-Venom/Eddie Brock: 5
- Scorpion: 5
- Black Cat: 5
- Shocker: 5
- Electro: 5
- Carnage: 4
- Doctor Octopus: 4
- Kraven the Hunter: 4
- Mysterio: 3
- Sandman: 3
- The Lizard: 3
- Vulture: 3
- Green Goblin/Norman Osborn: 3
- Kingpin: 3
- Hammerhead: 2
- Calypso: 2
- Green Goblin/Harry Osborn: 2
- Wolverine: 2
- Silver Sable: 1+1
- The Beetle: 1+1
- Morbius: 1
- Shriek: 1
- Boomerang: 1
- Juggernaut: 1
- Deadpool: 1
- Hobgoblin: 1
- Vermin: 1
- Iguana: 1
- Alistaire Smythe: 1
- Chameleon: 1
- Luke Carlyle: 1
- The Tinkerer: 0+1
It is possible I missed some appearances or otherwise made a mistake, but I think this is fairly accurate.
The biggest notable omissions is probably Hydro-Man. It's also notable that characters such as Hobgoblin and Chameleon only have one appearance each.