Introduction: Myocardial infarction (MI) is a life-threatening disease that occurs due to a mismatch between coronary blood supply and myocardial demands followed by blood, oxygen, and nutrients deprivation to the Cardiomyocytes. SuchThis disease is associated with excessive reactive oxygen species (ROS) release that damages the CardiomyocytesCardiomyocytes' membrane and subcellular structures with subsequent induction of inflammation and eventually apoptosis. One of the affected vital subcellular structures is the cardiac mitochondrial DNA (mtDNA). The present study used an isoproterenol (ISP)-induced MI rat model to investigate the role of the main active constituent of Nigella Sativa seeds, thymoquinone (TQ), in protecting the cardiac mtDNA content and ameliorating the oxidative stress, inflammation, and apoptosis. Methods: Rats in (TQ+ISP) group were pre-treated with TQ (20mg/kg/day) for 21 days before the MI induction using ISP (85mg/kg/day). In addition, negative control and ISP groups were included in the study for results comparison. Eventually, histopathologicalHistopathological examination was performed and serum cardiac parameters (cTnI and LDH) were assessed. Moreover, and mtDNA content, oxidative stress parameters (MDA, GSH, SOD, GPx, and CAT), inflammatory mediators (IL-6, IL-1β, and TNF-α), and apoptosis markers (BAX, Bcl2, and caspase-3) were detected. Results: TQ pretreatment showeddemonstrated a reverse of the infarction morphological changes in (TQ+ISP) group. Furthermore, pre and co-treatment with TQ caused a significant decrease in serum cardiac markers, inflammatory markers, significant attenuation in the apoptosis process, and preservation of the cardiac mtDNA content. Conclusion: TQ has a preserving impact on the cardiac mtDNA content in addition to its powerful antioxidant, anti-inflammatory, and anti-apoptotic action cardioprotective agentagents.