彼得·帕克 和他的大学科学教授柯特·康纳斯博士探索了新基因学作为肢体再生方法的方法。 但在对自己进行实验时，康纳斯不小心变成了蜥蜴，这是一种半人半爬行动物的怪物，它恐吓曼哈顿，随后绑架了自己的妻子。 蜘蛛侠的任务更加艰巨，因为他不想伤害他知道被困在里面的好朋友……但他必须阻止失控的非人野兽。

诺曼·奥斯本聘请斯宾塞·斯迈思设计一台能够摧毁蜘蛛侠的机器——黑寡妇蜘蛛杀手。 但是当闪电汤普森穿上蜘蛛侠服装作为派对噱头时，致命机器发现了他，他代替彼得被带走了。

斯宾塞·斯迈思死后，他的儿子阿利斯泰尔求助于金并帮助他报复蜘蛛侠和其他他认为应对父亲之死负责的人。 他俘虏了埃迪·布洛克、J·乔纳·詹姆森、诺曼·奥斯本和闪电汤普森。 然后，他将目光转向蜘蛛侠，并派出三名杀手追捕他。 蜘蛛侠在与他们单独作战时只能设法坚守阵地。 然而，当他们组合成超级杀手并将他带到斯迈思时，他就没那么幸运了。

奥托奥克塔维斯博士在一次实验室事故中与他的机械臂融合在一起，成为了疯狂的章鱼博士。 然后，他为哈代基金会给他造成的屈辱寻求报复。 他绑架了费利西亚并勒索了她的赎金。 因此，蜘蛛侠又一次出手相救。

J. Jonah Jameson hires Dr. Farley Stillwell who is a raining genius in genetic engineering, to make a new super hero that can match and defeat Spider-Man. With a willing participant, Mac Gargain, they engineer the DNA of a scorpion and him together to form a new hero, The Scorpion. However, when Mac thinks they have turned him into a freak, hero turns to monster and he goes on a rampage. Spider-Man is then forced to step in to stop creation from killing creator.

罗比为彼得安排了一位名叫玛丽亚·克劳福德博士的研究员的工作面试。 然而，当他到达她的实验室时，他发现她正遭到一个自称为猎人克莱文的怪物的袭击。 蜘蛛侠带领克劳福德离开克莱文来到屋顶。 在那里，克劳福德告诉蜘蛛侠，克莱文并不总是那样，事实上她爱他就像谢尔盖克拉文诺夫一样。 克劳福德讲述了多年前的一次事故，如果她没有使用一种危险的药水救了他的命，并赋予他动物的力量和本能，但却让他发疯，谢尔盖可能会丧命。 与此同时，克莱文在她的公寓寻找克劳福德时，得知她与罗比的关系并绑架了他。 然后他威胁说，如果蜘蛛侠不和克劳福德一起来找他，他就会杀死罗比。

A space shuttle piloted by J.J.'s son, John Jameson, crash lands on the Tri-Borough Bridge carrying two strange meteors -- One that could provide a new nuclear fuel and one that contains an alien substance. The first is stolen by the Kingpin's henchman, the Rhino, just as Spider-Man arrives to save Jameson and the co-pilots. When the Rhino sends the aircraft off the bridge, Spider-Man emerges from the water with a dark stain covering his costume. Exhausted, he heads for home, only to awaken hanging from a skyscraper, now clothed in a black version of his costume.

Spider-Man goes after Eddie Brock for labeling him as he is now being stalked by bounty hunters at every turn. After being reassured by Mary Jane that he has changed, he seeks help from Dr. Curt Connors who discovers that the suit is alive!! And Peter is the host organism!

The Kingpin is furious at Spidey and sends the Shocker and later the Rhino out to finish him off with the aid of another new invention of Alistair Smythe. Meanwhile, Eddie Brock, as the symbiote creature Venom, is busy playing cat and mouse with Spider-Man, intruding on Peter's private life and making it clear he knows the web-slinger's secret identity. Peter soon realizes that the pumped-up Eddie as Venom is stronger than he is. Worse than that: Spidey's spider-sense doesn't work around him!

Norman Osborn hires hitman and thief, the Hobgoblin, to take out Osborn's "silent" partner, Wilson Fisk (the Kingpin). When he fails in the attempt, the Hobgoblin changes sides, kidnapping Norman's son, Harry. Harry just happens to be best friends with Peter Parker.

When Aunt May is slightly injured by one of the Hobgoblin's pumpkin bombs during the kidnapping of Harry, Spider-Man enters the fray with a vengeance. After several battles, the trail leads Peter to Oscorp and Harry's own father. For Peter, rescuing his friend is easy compared to explaining to Harry that his own father is involved.

An international spy and hitman known as the Chameleon plans to assassinate two major diplomats on the brink of signing the peace proposal of the century. S.H.I.E.L.D. director Nick Fury solicits J. Jonah Jameson's assistance in an effort to prevent the "human shape-shifter" from derailing the proposal signing. But only Spider-Man with his super keen "spider-sense" can discern who is really whom and stop the criminal before it's too late.