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To evaluate the rate of presumed endophthalmitis (EO) after intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections in three European hospitals performed in an operation room (OR) under sterile conditions.A retrospective multicenter study between 2003 and 2016 at three European sites, City Hospital Triemli Zurich, Switzerland (CHT), Zealand University Hospital Roskilde, Denmark (ZUH) and University Clinic Bern, Switzerland (UCB). Intravitreal injection (IVI) database of each department was reviewed. All anti-vascular endothelial growth factor injections were performed using a standardized sterile technique in an operation room. Injection protocols were similar between the three sites. No preinjection antibiotics were given. Postoperative antibiotics varied among sites.A total of 134,701 intravitreal injections were performed at the 3 sites between 2003 and 2016. Ten cases of presumed endophthalmitis were documented: 4 in 50,721 at CHT (95% CI: 0.0071–0.0087%), 2 in 44,666 at ZUH (95% CI: 0.0039–0.0051%), and 4 in 39,314 at UCB (95% CI: 0.0092–0.011%). This results in one case in 13,470 intravitreal injections and a combined incidence of 0.0074% per injection (95% CI: 0.0070–0.0078%). Positive cultures were found in 4 out of 10 presumed endophthalmitis cases.The standardized sterile technique in an operation room with laminar airflow showed very low rates of endophthalmitis at three European sites.