Objective: Oxidative stress can cause DNA damage and apoptosis, and leads to cardiovascular disease. This study aims to evaluate the effect of aerobic exercise combined with consumption of hydroalcoholic extract of tribulus terrestris on mitochondrial oxidative stress markers in heart tissue of rats poisoned with hydrogen peroxide. Methods: This is an experimental study conducted on 42 male Wistar rats divided randomly into seven groups of Control (poisoned without supplementation), Aerobic Exercise, Aerobic Exercise + Supplementation with 5mg/kg extract, Aerobic Exercise + Supplementation with 10 mg/kg extract, Supplementation with 5mg/kg extract, Supplementation with 10mg/kg extract, and healthy control. All groups received hydrogen peroxide (100 mg/kg body weight) for 14 days intraperitoneally. The rats in supplementation groups received hydroalcoholic extract of tribulus terrestris with doses of 5 and 10 mg/kg of body weight by gavage. Aerobic exercise was performed on a treadmill at a speed of 23 m/min for 8 weeks, 5 days per week, each for 30 min. Twenty-four hours after the last exercise session, the heart tissues of rats were collected. Data were analyzed by independent t-test, two-way ANOVA, and Bonferroni post hoc test considering a significance level of P<0.05. Results: Consumption of tribulus terrestris extracts alone and in combination with aerobic exercise led to a significant increase in the levels of O6-methylguanine-DNA-methyl transferase, prooxidants-antioxidant balance, and cytochrome C oxidase, and a significant decrease in adenosine triphosphate and malondialdehyde levels compared to the control group (P<0.05). Conclusion: It seems that regular aerobic exercise and consumption of various doses of tribulus terrestris extract is a moderating factor in mitochondrial biogenesis, and is effective in reducing DNA damage in the heart tissue of rats. Lower dosage of tribulus terrestris extract has more benefits.