I would say that Doc Ock and the Green Goblin are tied as Spidey's top villains historically, while Venom and the Kingpin are both very interesting for their own reasons as well. Here is what I find so interesting about each as he matches up with the Web Slinger:
1) Dr. Octopus is a genius and a mental match for Peter Parker.
2) The Green Goblin is straight-up insane, making him incredibly unpredictable.
3) Venom is a physical match for Spidey, with the distinct advantage of not setting off his spider sense.
4) The Kingpin plays the role of puppet-master, often the driving force behind everything. His influence is wide-spread.
My final opinion is that it's hard to beat what the Green Goblin did in killing Gwen. Gun to my head, I say the Goblin wins, but it is fun to discuss!