Purpose Studying the effect of Mesna on middle ear otitis media and cholesteatoma induced by propylene glycol on an experimental animal model. Methods The study was designed to consist of sixteen Wistar albino rats, their right ears being the control group and left ears being the experiment group. %50 propylene glycol, gentamicinsulfate and physiologic salt water were applied to the right ear and %50 propylene glycol, gentamicinsulfate and %20 Mesna were administered to the left ear through intratympanic injections on days 1, 3, 8, 15 and 21. The rats were sacrificed 45 days after the first injection and underwent histopathological examination. Results It was seen that cholesteatoma and fibrosis were less common in the experiment group in microscopic evaluation. A statistically significant decrease was observed when the average and maximum thicknesses of the tympanic membranes and the minimum thicknesses of the tympanic bulla of the control group and the experiment group were compared. (p< 0.05) Conclusion In the experimental cholesteatoma model created in rats, no statistical significance was observed, indicating that Mesna, which was applied intratympanically, completely prevented the formation of cholesteatoma. However, it was found that the prevalence of cholesteatoma formation was microscopically less in the experimental group.