This study determined the preventive effect of melatonin on the occurrence of experimentally-induced myringosclerosis of the tympanic membrane (TM). Twenty Wistar albino-type rats weighing approximately 300 g each were randomly separated into two groups and myringotomized on the left TMs: group 1 rats (n=6) received intraperitoneal melatonin injections 10 mg/kg/day whereas group 2 rats (n=12) were treated with physiological serum only. The remaining two rats were served as the control group for histological comparison and standardization. After 15 days of treatment, myringotomized membranes were examined by otomicroscopy and harvested for histopathological evaluation. The functional effect of myringosclerotic plaques in the TMs of the two groups were compared with tympanometric measurements. Tympanic membranes in group 2 revealed extensive myringosclerotic plaques, on the other hand, TMs in group 1 showed faint or no existence of myringosclerosis. The mean magnitude of the maximum admittance from group 2 measured by tympanometry reduced to about 40% of the values obtained from group 1 (Z=-2,067, p=0.041). The mean magnitude of the maximum admittance from melatonin group was very close to the mean tympanometric value of non-myringotomized Wistar albino rats, demonstrating a functional outcome. The occurrence of myringosclerosis following experimental myringotomy can be hindered by systemic melatonin treatment.