Cantharellus miomboensis is a new basidiomycete fungus recently found in Miombo woodlands in Tanzania. In this study, crude extract was prepared from fruiting bodies of C. miomboensis and was in vitro screened for its cytotoxicity using Tetrazolium salt (3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) in human cell lines namely; Hepatocellular carcinoma (HepG2), Human non-small cell lung carcinoma (H157) and Human colon adenocarcinoma (HT.29). Thereafter, pro-apoptotic effects were determined using biochemical changes in apoptotic cells. These included externalization of phospholipid phosphatidylserine (PS) using APO Percentage dye by flow cytometry and depolarization of mitochondrial membrane potential using Tetramethyl rhodamine ethyl ester perchlorate (TMRE) assay. The test extract was found to induce dose dependent PS externalization on human cell lines when treated with various concentrations (1 - 5 mg/ml) and completely depolarized the mitochondrial membrane potential after 6 hours on HepG2 cell line. When the extract was examined for ROS production using 2’,7’- dichlorofluorescin diacetate (DCFH-DA) staining, there was no ROS generation found in HepG2 cells. It is therefore concluded that C. miomboensis extract is able to induce apoptosis in HepG2 cells and PS externalization and loss of mitochondrial membrane potential in HepG2 cells appear to be independent of ROS production.