In Brief Purpose: To investigate the bactericidal effect of 0.025% povidone–iodine in Balanced Salt Solution PLUS (0.025% PI–BSS PLUS) and its use in vitrectomy for postoperative endophthalmitis. Methods: First, an experimental laboratory model using Staphylococcus aureus was used to evaluate the bactericidal effect of PI–BSS PLUS. Next, in a case series of 4 eyes with postoperative endophthalmitis, vitrectomy using 0.025% PI–BSS PLUS as irrigation solution was conducted, followed by postoperative intravitreal and intravenous antibiotics. Results: In in vitro study, PI at concentrations of 0.01% and above in BSS PLUS exhibited marked bactericidal effect after 15 seconds of exposure. Bactericidal effect of 0.025% PI–BSS PLUS was maintained at room temperature storage for 15 minutes but was attenuated after 30 minutes. Among 4 eyes that underwent vitrectomy using 0.025% PI–BSS PLUS, coagulase-negative Staphylococcus sp. was isolated in 1 eye at the beginning but not at completion of surgery. In all four eyes, endophthalmitis was resolved with no adverse events. Ocular toxicity was not observed. Conclusion: The 0.025% PI–BSS PLUS is bactericidal and nontoxic when used as irrigation solution in vitrectomy. In 4 cases of postoperative endophthalmitis, vitrectomy using 0.025% PI–BSS PLUS followed by postoperative antibiotics resolved endophthalmitis. When used within 15 minutes of preparation, 0.025% povidone–iodine in Balanced Salt Solution PLUS was markedly bactericidal after 15 seconds of exposure. In 4 cases of postoperative endophthalmitis, vitrectomy using 0.025% povidone–iodine in BSS PLUS as irrigation solution followed by postoperative antibiotics resolved endophthalmitis, without ocular toxicity or adverse events.