PURPOSE: Routine use of face masks for patients and physicians during intravitreal anti-vascular endothelial growth factor (VEGF) injections has increased with the emergence of the coronavirus disease 2019 pandemic. This study evaluates the impact of universal face mask use on rates and outcomes of post-injection endophthalmitis (PIE). DESIGN: Retrospective, multicenter, comparative cohort study. PARTICIPANTS: Eyes receiving intravitreal anti-VEGF injections from October 1, 2019, to July 31, 2020, at 12 centers. METHODS: Cases were divided into a "no face mask" group if no face masks were worn by the physician or patient during intravitreal injections or a "universal face mask" group if face masks were worn by the physician, ancillary staff, and patient during intravitreal injections. MAIN OUTCOME MEASURES: Rate of endophthalmitis, microbial spectrum, and visual acuity (VA). RESULTS: Of 505 968 intravitreal injections administered in 110 547 eyes, 85 of 294 514 (0.0289%; 1 in 3464 injections) cases of presumed endophthalmitis occurred in the "no face mask" group, and 45 of 211 454 (0.0213%; 1 in 4699) cases occurred in the "universal face mask" group (odds ratio [OR], 0.74; 95% confidence interval [CI], 0.51-1.18; P = 0.097). In the "no face mask" group, there were 27 cases (0.0092%; 1 in 10 908 injections) of culture-positive endophthalmitis compared with 9 cases (0.004%; 1 in 23 494) in the "universal face mask" group (OR, 0.46; 95% CI, 0.22-0.99; P = 0.041). Three cases of oral flora-associated endophthalmitis occurred in the "no face mask" group (0.001%; 1 in 98 171 injections) compared with 1 (0.0005%; 1 in 211 454) in the "universal face mask" group (P = 0.645). Patients presented a mean (range) 4.9 (1-30) days after the causative injection, and mean logarithm of the minimum angle of resolution (logMAR) VA at endophthalmitis presentation was 2.04 (~20/2200) for "no face mask" group compared with 1.65 (~20/900) for the "universal face mask" group (P = 0.022), although no difference was observed 3 months after treatment (P = 0.764). CONCLUSIONS: In a large, multicenter, retrospective study, physician and patient face mask use during intravitreal anti-VEGF injections did not alter the risk of presumed acute-onset bacterial endophthalmitis, but there was a reduced rate of culture-positive endophthalmitis. Three months after presentation, there was no difference in VA between the groups.