The unconventional superconductivity in Sr2RuO4 is infamously susceptible to suppression by small levels of disorder such that it has been most commonly studied in extremely high-purity bulk crystals. Here, we harness local structural and spectroscopic scanning transmission electron microscopy measurements in epitaxial thin films of Sr2RuO4 to disentangle the impact of different types of crystalline disorder on superconductivity. We find that cation off-stoichiometry during growth gives rise to two distinct types of disorder: mixed-phase structural inclusions that accommodate excess ruthenium and ruthenium vacancies when the growth is ruthenium-deficient. Several superconducting films host mixed-phase intergrowths, suggesting this microstructural disorder has relatively little impact on superconductivity. In a non-superconducting film, on the other hand, we measure a high density of ruthenium-vacancies (∼14%) with no significant reduction in the crystallinity of the film. The results suggest that ruthenium vacancy disorder, which is hidden to many structural probes, plays an important role in suppressing superconductivity. We discuss the broader implications of our findings to guide the future synthesis of this and other layered systems. © 2022 Author(s).