Background: Previous studies have reported the antifertility activities of sulfonamides. This study was designed to evaluate the effects of co-trimoxazole and its co-administration with folic acid on ovarian tissue in female rats.Methods: A total of 54 rats were randomly divided into 9 groups (n=6). Group I served as the control and group II (vehicle) received saline. Other groups, III to IX, received co-trimoxazole (30, 60, and 120 mg/kg; i.p.), folic acid (1 mg/kg; i.p.) or their combination for 14 days, respectively. The oocytes were obtained from each group at the end of the 14th days and scored for maturational status as germinal vesicle (GV), metaphase I (MI), or metaphase II (MII). The number of primordial follicle (PrF), primary follicle (PF), and secondary follicle in formalin-fixed ovaries were counted under light microscopy. The data were analyzed by one-way ANOVA followed by post-hoc Dunnet test using SPSS statistical software (version 17.0). Results were considered statistically significant at P<0.05.Results: Co-trimoxazole (60 and 120 mg/kg) treatment for 14 days caused a significant decrease in the number of GV (P=0.02, P<0.001), MI and MII (P=0.03, P<0.001), a significant increase in structural abnormalities, including PrF, PF and secondary follicle (P<0.001) as well as congestion, inflammation and necrosis of ovarian tissue compared to the vehicle group. Folic acid co-administration with co-trimoxazole reversed partially all these parameters compared to the co-trimoxazole group (P<0.001).Conclusion: The data showed the adverse effects of co-trimoxazole on the ovarian maturational status and tissue structure which was reversed partially by folic acid co-administration in rats.