To compare the rates and outcomes of endophthalmitis after intravitreal injections for patients pre-treated with 5% povidone-iodine (PI) and 0.05% chlorhexidine (CHX). A retrospective, single center, comparative cohort study. A total of 8,686 patients were included in the study and a total of 67,695 anti-VEGF injections were performed from January 1, 2019, to November 30, 2021. A private retina practice of 13 retina specialists in Colorado. Using electronic health records and billing code data, patients with pre-injection preparation with PI or CHX were compiled retrospectively. Use of prefilled (PFS) versus non-prefilled syringes was also tabulated. Patients with post-injection endophthalmitis (PIE) were identified among the two groups. PIE is defined as an inflammation of the eye from infectious agents following an intravitreal injection. LogMAR visual acuity was determined at the time of causative anti-VEGF injection, endophthalmitis presentation, and 3-month follow-up. A total of 67,695 intravitreal injections (IVI) were administered by 13 retina specialists during the study period. 13 of 32,802 (0.0396%; 1 in 2,523 injections) cases of presumed endophthalmitis occurred in the PI group, and 9 of 34,893 (0.0258%; 1 in 3,877 injections) cases in the CHX group (p=0.395; Table 1). For the PI group, there were 2 culture-positive endophthalmitis cases (0.0061%, 1 in 16,401), compared to 2 cases in the CHX group (0.00573%, 1 in 17,447) (p=1.000; Table 1). No significant difference was observed in the average logMAR visual acuity of PI and CHX at causative injection (p=0.3851), endophthalmitis encounter (p=0.7718), and 3-month follow-up (p=0.6152; Table 2). A significant reduction in presumed endophthalmitis (p=0.0445) and culture-positive cases (p=0.0130) was present for prefilled IVI compared to non-prefilled injections. No significant difference in the rate of endophthalmitis or in visual outcomes were found in the CHX group compared to the PI group. Prefilled syringes were associated with a significant reduction in endophthalmitis rate. Further multicenter studies are needed to validate the efficacy and safety of CHX compared to PI for intravitreal injection preparation.