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The study evaluated the effects of berberine on sexual activity and crucial biomolecules linked to penile function in diabetic rats. Sixty-three (63) adult male rats were used and divided into nine groups (n = 7). Group I received 0.1 M citrate buffer at pH 4.5 orally as normal control. Group II received 5.0 mg/kg sildenafil citrate orally. Groups III and IV rats received 50.0 mg/kg and 100.0 mg/kg of berberine orally, respectively. Group V rats were induced with STZ intraperitonially (50 mg/kg b.w.t.) (diabetic control rats). Group VI diabetic erectile dysfunctional (DED) rats received sildenafil citrate orally. Groups VII and VIII DED rats received 50.0 or 100 mg/kg of berberine orally, respectively. Group IX DED rats received 25 mg/kg of acarbose. The result revealed that blood glucose level was reduced in diabetic groups treated with berberine, sildenafil citrate, and acarbose when compared to the diabetic control group. In addition, mounting and intromission numbers as well as antioxidant biomarkers/status were enhanced in normal rats as well as in berberine-treated diabetic rats when compared to diabetic control rat group. Conversely, α-amylase and α-glucosidase activities were significantly (P < 0.05) elevated in diabetic rat with erectile dysfunction in comparison to the normal rats and berberine-treated diabetic rats. In conclusion, berberine could represent a potential therapeutic agent for the treatment of diabetes-induced ED.