Background: Traditional medicine has long utilized herbal and animal-derived materials worldwide, and traditional formulations for the management of male sexual dysfunction continue to be actively investigated. This study aimed to investigate the chemical composition and antioxidant effects of Anti-Impotence-4, an herbal–animal formulation, in an experimental model of chronic stress-induced erectile dysfunction (ED). Aim: To investigate the bioactive compounds present in the plant- and animal-derived formulation Anti-Impotence-4 and its effects against oxidative stress. Materials and Methods: Fifty-eight male Wistar rats were randomly divided into six groups, including healthy control, disease control, reference, and treatment groups. Chronic stress was induced once daily for 56 days using a random stress protocol, as described by Peng Zou (2019). Oxidative stress parameters were determined using ELISA. The chemical constituents of the methanolic extract of Anti-Impotence-4 were analyzed using ultra-performance liquid chromatogra phy–mass spectrometry (UPLC–MS) in both positive and negative ionization modes. Results: In an experimental model of erectile dysfunction induced by chronic stress, treatment with Anti-Impotence-4 led to a dose-dependent attenuation of oxidative damage. Specifically, levels of the DNA oxidation marker 8-hydroxy-2′-de oxyguanosine (8-OHdG) were reduced by 13.3–32.5% following administration of Anti-Impotence-4 at doses of 42, 84, and 210 mg/kg. Likewise, malondialdehyde (MDA) concentrations decreased by 7.9–23.7% at therapeutic doses (p<0.05). In contrast, antioxidant defense mechanisms were markedly enhanced, as evidenced by significant increases in superoxide dismutase (SOD) activity (34.1%, 38.1%, and 44.6%, respectively; p<0.05) and glutathione (GSH) levels, which rose by 37.0–55.4%. Biological substances identified a total of 10 compounds, including six amino acids, flavo noids, and iridoids, in the positive ion mode. In negative-ion mode, 18 bioactive compounds were detected, including amino acids, carbohydrates, organic acids, fatty acids, flavonoids, and phenolic compounds. Conclusion Phytochemical analysis of the methanolic extract of the Anti-Impotence-4 formulation demonstrated the presence of diverse bioactive constituents, including amino acids, purine derivatives, nucleosides, carbohydrates, organic and fatty acids, flavonoids, phenolic compounds, and iridoids. In a chronic stress-induced erectile dysfunction model, treatment with Anti-Impotence-4 significantly enhanced antioxidant defense by increasing superoxide dismutase and glutathione levels, while markedly reducing oxidative stress markers such as 8-hydroxy-2′-deoxyguanosine and malond ialdehe indicating significant antioxidant and protective effects.