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The aim of this study was to evaluate the comparison of the efficacy of royal jelly in a guinea pig tympanic membrane perforation model with untreated and control groups.Prospective study using 16 adult guinea pigs.Each animal underwent subtotal removal of the tympanic membrane in one ear. Animals were divided into two groups of eight animals each. Group A was treated with topical royal jelly, and group B was treated with saline. All opposite right ears were used as the control group. Examination was done by otoendoscopy on days 10, 12, and 14. The healing process was observed under the microscope and photographed for 3 months.There was no significant difference between group A and group B in the incidence of perforation closure. However, after 3 months, the mean thickness of the tympanic membranes was 0.11, 0.07, and 0.08 mm, respectively, in group A, group B, and the control. These results showed a statistically significant difference. Histologically, the tympanic membranes of group A showed marked fibroblastic orientation and well-organized connective tissue. Application of royal jelly improved the success rate of closure, as shown in the present study.This is the first study to investigate the effectiveness of royal jelly in enhancing healing in tympanic membrane perforations. Our study suggests that royal jelly is effective in increasing healing of tympanic membrane perforations in guinea pigs.