Cytotoxic activity profiling of the n-hexane extract from the fruit pulp of Ginkgo biloba L. (Gingkgoaceae) led to the purification of 8 compounds. HPLC based extract profiling identified a cytotoxic alkenylresorcinol. The extract was further separated by time-based fractionation in a gradient HPLC condition, and cytotoxicity of each fraction was evaluated using 293, B16F10, BJAB, and HCT116 cancer cells in vitro. Bilobol was isolated as an active constituent by semi-preparative HPLC and showed significant cytotoxic activities against the cancer cells in a dose-dependent manner at the concentrations of 15.0 ~ 50 μg/mL. The western blotting analysis showed that this compound increased expression of active caspase-3 and active caspase-8 in HCT116 human colon cancer cells, indicating that this compound causes apoptotic cell death in a dose-dependent manner. The results suggest that bilobol is the active component from fruit pulp of Ginkgo Biloba L. that can induce apoptotic cell death in various cancer cells.