Background Esculetin is a natural coumarin derivative from various plants with multiple pharmacological effects. Hence, the present study was undertaken to explore the cardio protective potential of esculetin against isoproterenol induced myocardial toxicity in rats. Methods The treatment schedule was fixed for 28 days and the rats were divided into five groups of six each. Rats of group I received the normal saline and served as normal control, group II was received ISO (100 mg/kg body weight) for last two consecutive days of the study and served as disease control. Groups III and IV received esculetin 10 and 20 mg/kg body weight respectively once a day per oral for 28 days along with ISO for last two consecutive days of the study. Cardiac biomarkers such as CK-MB and LDH, membrane bound Na+ /K+ ATPases activity, myocardial lysosomal enzymes activity and tissue antioxidants status were estimated in the heart tissue samples. The histopathological changes in the myocardium were also assessed. Further, DPPH assay was done to evaluate the free radicals scavenging potential of esculetin. Cytoxicity assay, intracellular ROS levels by DCFDA assay and m-RNA expression of TNF-α, IL-6 and NF-κB by quantitative RT-PCR in H9c2 cell lines. Results The increased levels of CK-MB, LDH, LPO, myocardial lysosomal enzymes and membrane bound Na+ /K+ ATPase levels by ISO administration was significantly increased with concomitant decrease in tissue antioxidant enzymes such as GSH, Catalase, and SOD. Pre-treatment with esculetin for 28 days has significantly decreased the levels of cardiac bio-markers, lysosomal enzymes, membrane bound Na+ /K+ ATPase levels as well as Lipid peroxides which is in contrary to the ISO group. Amelioration of the antioxidant levels were also found in esculetin treated groups. Histopathological examination of heart reveals that myocardial degeneration, mononuclear cell infiltration was noticed in ISO treated rats, whereas the same was restored with esculetin treatment. In H9C2 cell lines esculetin could effectively reduced intracellular ROS inhibition and m-RNA expression of pro-inflammatory cytokines including TNF-α, IL-6 and NF-κB to prevent apoptosis or cell necrosis. Conclusion The study provides the evidence of cardioprotective potentials of esculetin against isoproterenol induced myocardial infarction by antioxidant and myocardial membrane stabilization along with in vitro protection from arsenic induced ROS cell necrosis or apoptosis in H9C2 cells.