"Agents of S.H.I.E.L.D." offers a positive portrayal of LGBTQ+ characters, primarily through Joey Gutierrez, an openly gay hero depicted with dignity and agency. Minor representation also exists with Deke Shaw. The show avoids negative stereotypes, presenting queer identity as a natural aspect of its characters without it being a source of conflict or mockery.

The show features several female characters, including Melinda May, Daisy Johnson, and Bobbi Morse, who are highly skilled in martial arts and hand-to-hand combat. They are frequently depicted winning physical fights against multiple male opponents, demonstrating superior combat abilities.

Daisy Johnson, a character canonically depicted as white in Marvel Comics, is portrayed by Chloe Bennet, who is biracial (Chinese-American and white), in the series. This constitutes a race swap.