Background. Antrodia cinnamomea is an edible fungus commonly used in Asia as a well-known medicinal herb capable of treating drug intoxication and liver cancer. Methods. This study evaluated the anticancer activity of its biotechnological product, mycelial fermentation broth (AC-MFB) on hepatocellular carcinoma (HCC) by tetrazolium-based colorimetric assay in vitro and syngeneic Balb/c 1MEA.7R.1 tumor implantation model in vivo. Given that cancer stem cell characteristics, such as angiogenesis, invasiveness, and migration, are known to cause recurrence, we further evaluated the effect of AC-MFB on cellular viability inhibition of HCC cells, angiogenic activity and migration of endothelial cells, and the release of proangiogenic factors from HCC cells. Results. We found that AC-MFB markedly inhibited the growth of HCC without hepatic enzyme abnormality. This anti-HCC activity was validated by growth-inhibitory effects on both cultured murine 1MEA.7R.1 and human HA22T/VGH HCC cells. For cancer stem cell characteristics, AC-MFB inhibited the cellular viability, migration, and tube formation activity of EA. hy926 and SVEC4-10 endothelial cells. Production of extracellular vascular endothelial growth factor and intracellular hypoxia-inducible factor-1 alpha from HCC cells was suppressed by AC-MFB. Conclusion. Antrodia cinnamomea could inhibit the growth and cancer stem cell characteristics of HCC cells.