BACKGROUND: This study examines the impact of commonly used disinfectants on hearing and the structural integrity of the inner ear. OBJECTIVE: To evaluate the effects of indophor, alcohol, and chlorhexidine on auditory function and cochlear morphology in mice. METHODS: Sixty-four mice were evenly divided into four groups: saline, indophor, alcohol, and chlorhexidine. Each mouse received a 25 μL injection of the designated liquid into the left ear, while the right ear was remained untreated. Auditory brainstem response (ABR) testing was performed at three intervals: before injection, 24 h post-injection, and one week post-injection. Cochlear tissues from both ears were analyzed using immunofluorescence and scanning electron microscopy to observe morphological changes. RESULTS: Chlorhexidine and alcohol significantly increased ABR thresholds in the left ear 24 h and one week post-injection (p <
0.001). Chlorhexidine caused severe damage to outer hair cells, inner hair cells, and spiral ganglion cells, while alcohol had a milder effect. Iodophor mainly affected outer hair cells, with partial recovery after seven days. In the right ear, only chlorhexidine caused a sustained ABR threshold increase (p <
0.001) and severe damage to outer and inner hair cells. Both alcohol and chlorhexidine induced demyelination of spiral ganglion cells in the left ear, but recovery was seen only in the alcohol group after one week. CONCLUSION: Indophor, alcohol, and chlorhexidine can all affect hearing, with alcohol and chlorhexidine causing more severe and lasting damage. These findings provide crucial insights for selecting disinfectants in ear surgeries. LEVEL OF EVIDENCE: NA Laryngoscope, 2025.