To investigate the temporal efficacy and sterility of povidone-iodine (PI) against bacteria associated with postinjection endophthalmitis in an in vitro study. A single PI bottle was opened and tested for sterility and antibacterial efficacy. Povidone-iodine from the open bottle was inoculated onto a blood agar plate and evaluated for growth 24 hours later to test sterility; this was repeated for five consecutive days. To test for antibacterial efficacy, PI was instilled onto the surface of plates of Staphylococcus , Streptococcus , Enterococcus , Pseudomonas , and Haemophilus species. The plates were then evaluated 24 hours later for a clear zone of inhibition (i.e., no growth) where the PI was placed. This process was repeated for five consecutive days. Sterility testing showed no growth of organisms on the blood agar plates for each of the five days of testing. For antibacterial efficacy testing, zones of inhibition of growth were seen on each plate of bacterial colonies only where PI was placed, for each of the five days of testing. In this study, PI from an open bottle maintained its sterility and antibacterial efficacy for five days. These data could help support repeated use of the same bottle, reducing waste and costs for retina practices.