Increased cell death of cardiomyocyte by oxidative stress is known to cause dysfunction of the heart. O. gratissimum is one of the more well-known medicinal plants among the Ocimum species and widely used in treatment of inflammatory diseases. In this study, we hypothesized that aqueous extract of O. gratissimum leaf (OGE) may protect myocardiac cell H9c2 from oxidative injury by hydrogen peroxide (H2O2). Our results revealed that OGE pretreatment dose-dependently protects H9c2 cells from cell death when exposed to H2O2. Additionally, DNA condensation induced by H2O2 was also reduced by OGE pretreatment, suggesting that Ocimum gratissimum extract may attenuate H2O2-induced chromosome damage. Further investigation showed that OGE pretreatment inhibited H2O2-induced activation of caspase-3 and caspase-9, as well as H2O2-induced upregulation of proapoptotic Apaf-1 and the release of cytosolic cytochrome c, but has little effect on the activation of caspase-8. Additionally, OGE pretreatment significantly upregulated Bcl-2 expression and Akt phosphorylation, and slightly affected the phosphorylation of mitogen-activated protein kinases including p38 MAPK and JNK. Taken together, our findings revealed that Ocimum gratissimum extract effectively inhibited the mitochondrial pathway and upregulated Bcl-2 expression, which may be important in protecting H9c2 cells from H2O2-induced cell death.