Gwen Stacy is already in Peter's lifeAt one point, Peter is seen chatting with an instant messenger client. His friend has a username called "GS122". Gwen Stacy originally died in Amazing Spiderman issue 121, implying that she would have eventually joined the series and survived.
If there was a second season, The Punisher would make an appearance.Thanks to Garth Ennis the character had really taken off, and with the Darker and Edgier storyline and New York wanting Spider-Man's head he would be a good way to not only kill off the original villains; forcing Peter to become Spider-Man again, but to resolve the character conflict at the end of the series.
Silver Sable may have survived and would have appeared in the second season.
The Green Goblin would have made an appearance as a hallucination.To Peter, out of guilt; or Harry, to tie-in with Spider-Man 2 (making the cartoon semi-canon with the movies).
If there was a second season, more characters from the movies would make appearances.
- Especially Aunt May, Uncle Ben, and Robbie Robertson. And Green Goblin, as aforementioned above.
The Lizard would have been revived and be cured of his mutation in the second season.As mentioned above, to make the cartoon semi-canon, as Curt Connors is alive and well.
Daredevil would have made an appearance in the second season.And be voiced by Ben Affleck, or a sound-alike.