Night at the Museum is a family-friendly adventure comedy that does not feature any identifiable LGBTQ+ characters or themes. The narrative centers on a security guard and the magical reanimation of museum exhibits, with no elements related to queer identity or experiences.

Night at the Museum does not feature any identifiable transsexual characters or themes. The film's narrative and character roster do not include any elements that would allow for an evaluation of transsexual portrayal.

The film features prominent female characters such as Sacagawea and Rebecca Hutman. However, neither character participates in direct physical combat. Sacagawea provides guidance, while Rebecca is a civilian. No female character is depicted defeating male opponents in close-quarters physical combat.

The film features historical figures and original characters. All historical figures, such as Theodore Roosevelt, Jedediah Smith, Octavius, Attila the Hun, and Sacagawea, are portrayed with their historically established genders. No characters established in prior canon or history have their gender changed.

The film features historical figures whose on-screen portrayals align with their documented race. Original characters created for the film do not have a prior canonical race to be swapped. No instances of race swapping were identified.