OBJECTIVE A paucity of data exists regarding the use of iodophores such as povidone-iodine (PVI) to disinfect water. We sought to determine a practical minimal disinfecting concentration of 10% PVI over different contact times and temperatures when added to water inoculated with E. coli. METHODS 1:100, 1:1,000, and 1:10,000 dilutions of 10% PVI were created. Escherichia coli was exposed to these dilutions for 5, 15, and 30 minutes at 10, 20, and 30°C. Bactericidal activity was neutralized with 0.5% sodium thiosulfate. Mean viable colony forming units (CFUs) was determined after triplicate plating on Luria-bertani agar and 24 hours of incubation at 37°C. Effective bactericidal activity was defined as a 5-log reduction. RESULTS Of the 200,000 E. coli plated, no CFUs were observed after exposure to the 1:100 dilution. After 5 minutes of contact time with the 1:1,000 dilution, at 10°C CFUs were too numerous to count (TNTC), at 20°C the mean CFU count was 92 (standard error ±11), and at 30°C the mean CFU count was 25 (standard error ±8). No CFUs were observed after 15 minutes of exposure to the 1:1,000 dilution across experimental temperatures. The 1:10,000 dilution always yielded CFU growth that was TNTC. CONCLUSIONS The lowest disinfecting concentration of 10% PVI was the 1:1,000 dilution at 15 minutes of contact time. This supports the use of PVI for water disinfection against E. coli, the organism most commonly responsible for traveler's diarrhea. Further studies may assess its effectiveness against more virulent water borne pathogens.