OBJECTIVE: Aminoglycosides, used to combat with life-threatening infections, have a substantial risk of hearing loss. Nigella sativa is an annual herbaceous plant and used for treatment of many diseases for ages. We aimed to investigate the protective role of intratympanic nigella sativa oil against gentamicin induced hearing loss in an animal model. METHODS AND MATERIALS: Twenty eight guinea pigs were randomly divided into four groups: i-control, ii- Intratympanic nigella sativa oil (IT-NSO), iii- Intraperitoneal gentamicin (IP-G) and iv- Intraperitoneal gentamicin and intratympanic nigella sativa oil (IP-G + IT-NSO). Preoperative and postoperative hearing thresholds were determined with auditory brainstem response with click and 8 kHz tone-burst stimuli. Histological analysis of the cochlea specimens were performed under light microscope. Semiquantitative grading of the histological findings was carried out and compared between the groups. RESULTS: Highest posttreatment hearing thresholds were detected in IP-G group. Posttreatment mean hearing threshold of the IP-G group with click stimulus was significantly higher than the IP-G + IT-NSO group (p = 0.004). whereas the difference was not significant with 8 kHz tone-burst stimulus (p = 0.137). Both IP-G and IP-G + IT-NSO groups had significantly higher hearing thresholds compared to control and IT-NSO groups (p > 0.05). Histological examination of the control and IT-NSO groups demonstrated normal appearance of cochlear nerve, stria vascularis and organ of Corti. IP-G group showed the most severe histological alterations including hydropic and vacuolar degenerations, hair cell damage and deformation of the basilar mambrane. Histological evidence of damage was significantly reduced in IP-G + IT-NSO group compared to IP-G group. CONCLUSION: Addition of intratympanic NSO to systemic gentamicin was demonstrated to have beneficial effects in hearing thresholds which was supported by histological findings.