The possible cochlear toxicity of topically applied povidone-iodine solution was investigated in guinea pigs by measuring transiently evoked otoacoustic emissions (TEOAEs). Seven adult pigmented guinea pigs (14 ears) with a normal acoustic pinna reflex were used. After the baseline TEOAE measurements, 0.1mL of povidone-iodine solution was applied transtympanically into the middle ear in the study group. Saline solution and gentamicin were used as negative and positive control, respectively. TEOAE measurements were repeated on the 10th day, and four weeks after the first application. Tympanometric measurements were also carried out for both ears of all animals at the end of the study. Responses in all ears, which had povidone-iodine applied to them, disappeared in a way similar to those recorded in gentamicin-treated ears. Tympanometric measurements at the end of the study showed ’type A’ tympanograms in all ears of all animals. The present study showed that povidone-iodine could have a cochleotoxic effect in guinea pigs. A study in humans with TEOAE before and after middle-ear surgery, where povidone-iodine can reach into the middle-ear cavity, may be useful for evaluating this result for clinical practice.