Previous in vitro studies in our laboratory demonstrated that ethyl acetate (P₂) and water- soluble (PS/PT1) fractionated leaf extracts of Ocimum gratissimum inhibit the proliferation of prostate cancer cells. It has been reported that the crude aqueous extract induces apoptosis in lung adenocarcinoma cells; however, the efficacy of the fractionated extracts against these cells remains unclear. In the present study, we hypothesized that the ability of the fractionated extracts to inhibit proliferation and induce apoptosis is associated with the activation of pro-apoptotic proteins and induction of DNA condensation in A549 cells. Ocimum gratissimum was cultivated and its leaves were harvested, extracted, and fractionated to produce fractions P₂ and PS/PT1. Anti-proliferative activity was assessed by direct cell count. For morphological characterization of apoptosis, 4′,6-diamidino-2-phenylindole staining was employed. Western blot analysis was performed to evaluate the apoptotic activity of the fractionated extracts. In data generated from anti-proliferation studies, P₂ significantly inhibited cell proliferation in a concentration-dependent manner; PS/PT1 elicited a decrease in the viability of cells, occurring at 500 µg/mL. 4′,6-diamidino-2-phenylindole staining revealed the induction of apoptosis, as evidenced by the formation of apoptotic bodies. Increased levels of pro-apoptotic proteins were observed as the concentrations of the fractionated extracts increased. These results suggest that fractionated leaf extracts of Ocimum gratissimum inhibit the proliferation and induce apoptosis of A549 cells.