To correlate the incidence of postoperative endophthalmitis with changes in the preoperative prophylaxis over a 20-year period. Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany. Retrospective chart review. Patients diagnosed with postoperative endophthalmitis from 1990 to 2009 after intraocular surgery performed at the same institution were included. Because of changes in the preoperative prophylaxis during the study period, 3 groups were formed for data analysis: Period 1 (1990 to 1992), no standardized prophylaxis regimen; period 2 (1993 to 1998), preoperative topical medication, povidone-iodine 10.0% periorbitally, and 1 drop of povidone-iodine 1.0% in the conjunctiva sac; and period 3 (1999 to 2009), similar to period 2 except with irrigation of the conjunctival sac with 10 mL of povidone-iodine 1.0%. The overall rate of postoperative endophthalmitis was 0.113% (77/68,323) for all intraocular surgeries. It decreased significantly from 0.291% (16/5505) in period 1 to 0.170% (33/19,413) in period 2 to 0.065% (28/43,405) in period 3 (P < .001). In cataract surgery, the overall rate of postoperative endophthalmitis was 0.125% (30/24,034). It decreased in each subsequent period, from 0.338% (9/2662) in period 1 to 0.224% (15/6696) in period 2 to 0.041% (6/14,676) in period 3 (P < .001). Coagulase-negative Staphylococcus was the most commonly isolated organism (47.4%). The rate of postoperative endophthalmitis decreased over a 20-year period at a single academic institution. Although multiple factors might have contributed to this decline, implementation of a preoperative prophylaxis protocol using copious povidone-iodine might have been the most important contributor. No author has a financial or proprietary interest in any material or method mentioned.