PURPOSE: To evaluate the ototoxicity of povidone iodine (PVP-I) at concentrations of 5% and 10% during tympanoplasty by assessing frequency-specific audiometric outcomes and graft success rates in a large cohort of patients. METHODS: This prospective, triple-blinded, randomized clinical trial enrolled 542 patients undergoing primary tympanoplasty. After excluding patients for inadequate follow-up, 204 ears received 10% PVP-I and 199 ears received 5% PVP-I. Audiometric evaluations, including bone conduction (BC), air conduction (AC), air-bone gap (ABG), and speech reception threshold (SRT), were conducted preoperatively and 12 months postoperatively. The primary outcome was the incidence of sensorineural hearing loss (SNHL), defined as an increase in BC threshold of more than 10 dB. Secondary outcomes included graft success rate and audiometric gains. RESULTS: The overall incidence of SNHL was 1.5% in the 10% PVP-I group and 1.0% in the 5% PVP-I group, with no significant difference (P = 1.000). In ears with preoperative mixed hearing loss, SNHL at 0.25 kHz was significantly higher in the 10% PVP-I group (28.9%) compared to the 5% PVP-I group (6.7%) (P = 0.002). Graft success rates were 94.1% and 97.0% in the 10% and 5% PVP-I groups, respectively (P = 0.164). No clinically significant differences in audiometric gains were observed between the groups. CONCLUSION: Both 5% and 10% PVP-I concentrations are safe for antiseptic preparation in tympanoplasty, as evidenced by low rates of SNHL and high graft success rates. These findings support the use of PVP-I in human otologic surgery without significant ototoxic risk.