Povidone-iodine is an effective broad-spectrum disinfectant with no reported toxicity to the cornea and conjunctiva when applied topically in single dose to the ocular surface. We challenged four strains of Neisseria gonorrhoeae, a clinical isolate of Chlamydia trachomatis, and one strain of herpes simplex virus type II with three different concentrations of povidone-iodine (5%, 1%, and 0.1%) for one minute. The challenge inoculum of Neisseria gonorrhoeae and herpes simplex virus type II were completely sterilized by all three solutions. The chlamydia titer was reduced by two log units at the 5% and 1% concentrations, but not the 0.1% concentration. Povidone-iodine may be of potential use in the prophylaxis of newborns against ophthalmia neonatorum.