Aunt May in the mainstream comics is usually depicted as having blonde hair before she went grey. Most modern productions that give her the Age Lift treatment (The Amazing Spider-Man Series, the Marvel Cinematic Universe, Spider-Man (PS4), etc.) tend to depict her with darker hair colors (reddish-brown, brown, or black), presumably to emphasize her and Peter as relatives.
Liz Allen who’s blonde in the comics, is brunette in The Spectacular Spider-Man, Marvel's Spider-Man and the MCU. The MCU example is due to Liz getting a Race Lift (having a black mother), while the cartoons likely just made Liz darker haired so she’d be distinct from Gwen Stacy.
Comic Books
Spidey: Flash has dark hair instead of his usual blond, and May also has dark hair instead of grey in School's Out. The Osborns are black haired instead of redheads.
Peter Parker has brown eyes in the comics; in the Sam Raimimovies, Tobey Maguire plays him, who has blue eyes. Although in the comics, they flip-flop between brown and blue so much eventually Marvel officially stated they were hazel as compromise.
The lenses on Spider-Man mask are traditionally plain white as seen in the comics, cartoons and MCU, while Raimi films makes them more realistically opaque like glass. The Amazing Spider-Man however instead has the lenses be gold. Although the sequel and No Way Home quickly ditches this look for wide pale lenses similar to the Raimi films.
Costume example with Norman as in the comics, he has an iconic evil green and purple colour scheme. In the Raimi film his goblin suit is instead all green. However Spider-Man: No Way Home masterfully manages to get in the comic colours, by having Norman wear a purple jumper over his green flight armor that rips away to reveal the green during his fight with MCU Spidey, making him look like his comic version.
In the comics Harry has the same green and purple costume as his father. In Spider-Man 3 he has a dark green and black outfit while in ASM2 his Goblin outfit is all green like the Raimi flight suit.
Doctor Octopus has a traditionally bright green and yellow outfit. In Spider-Man 2 he has a brown Badass Longcoat and following the film it became more common to see Otto in a trench coat in the comics. No Way Home gives him a green coat, but it’s much darker than the comic versions.
Jerk Jock Flash Thompson is black haired in the Raimi trilogy as opposed to the red or blonde hair of the comics; this was corrected in The Amazing Spider-Man where he's blonde.
The usually grey-haired Aunt May is blonde in Spider-Man: The Animated Series. Also, Eddie Brock has red hair rather than blonde.
A brunette during her tenure in the comic books, Sally Avril was turned into a blonde for The Spectacular Spider-Man, with her hair also going from being short and curly to long and straight.
Spider-Man villain Doctor Octopus is almost always portrayed as having short brown hair, but sports long black hair in the Ultimate Spider-Man (2012) animated series.
Carnage's human host Cletus Kasady is a redhead in the comics, but was depicted as blond in Spider-Man Unlimited.
Eddie Brock has brown hair instead of blonde hair.
J. Jonah Jameson has brown hair instead of either gray or black.
In Marvel's Spider-Man, Harry Osborn has black hair instead of either red-brown or blond like in other shows. Similarly, Alistair Smythe has ginger hair instead of brown. Likewise, Peter Parker has green eyes instead of black, blue, or brown. Others also appearing in this show include the Man-Wolf having dark gray fur and blue eyes, rather than white fur and red eyes, Liz Allan is usually portrayed as being a blonde, but this version portrays her a brunette and Aleksei Sytsevich, who is a redhead as opposed to being a brunette in the comics and most versions.
