Microbes have long been used for the synthesis of a variety of nanoparticles. Hepatocellular carcinoma (HCC) is the primary liver cancer and it is the second leading cause of cancer-related mortality worldwide. In this study, we have synthesized Enterococcus mediated gold nanoparticles (AuNPs) and investigated their cytotoxic potential against human hepatocellular cancer cell line (HepG2). AuNPs were synthesized using Enterococcus sp. RMAA. HepG2 cells were treated with different concentrations of AuNPs for 24 hours and cytotoxicity was analyzed by MTT ((4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. AuNPs induced reactive oxygen species expression was analyzed by 2',7'-dichlorodihydrofluorescein diacetate staining. Morphological changes related to apoptosis was analyzed by annexin V/propidium iodide staining. Protein expression of proliferating cell nuclear antigen (PCNA) was done by western blotting analysis. Bacterial-mediated AuNPs caused significant cytotoxicity in HepG2 cells. AuNPs treatment also caused the significant expression of ROS and morphological damage related to apoptosis. AuNPs treatments were responsible for the dislocation of cytochrome c from mitochondria to cytosol. The protein expression of PCNA was significantly decreased upon AuNPs treatment. These findings suggest that Enterococcus-mediated AuNPs can inhibit the proliferation of HepG2 cells via intracellular ROS mediated apoptosis, decreased PCNA expressions, and it may have the potential to treat HCC.