Ginkgo biloba extract EGb761 conveys an anticancer effect, but little is known regarding its role in hepatocellular carcinoma (HCC). Our study aims to determine the anticancer effect of EGb761 on HCC cell lines and clarify the underlying molecular mechanism. We explore biological functions of EGb761 in HCC using morphological observation, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cytotoxic analysis. We investigate the effects of EGb761 on proliferation and apoptosis of HCC cells using plate clone formation, proliferating cell nuclear antigen, and terminal deoxynucleotidyl transferase d-untranslated protein nick end labeling assays. Protein expressions of the NF-κB/p53 signal pathway were detected and identified using immunohistochemistry. The effect of EGb761 on the p53 signaling pathway was further confirmed by adding pifithrin (PFT)-α, an inhibitor of p53. We determine that EGb761 inhibits cell growth, reduces cell viability, and promotes apoptosis of HCC cells. In addition, EGb761 reduces proliferation and increases apoptosis of human hepatocellular carcinomas (HepG2) cells in a dose-dependent manner. We also find that EGb761 exerts an anticancer effect on HepG2 cells by activating p53 and inhibiting nuclear factor (NF)-κB signaling pathways. This study confirms that EGb761 inhibits proliferation and triggers apoptosis of HCC cells through the NF-κB/p53 signaling pathway.