Ethnopharmacological relevance: The rhizomes of Dioscorea membranacea Pierre (DM) have been used as ingredients in anticancer herbal formulations in Thai traditional medicine (TTM). Thus, the aim of this study was to investigate the active constituents of DM for cytotoxic activity in order to support its TTM use. Materials and methods: A bioassay-guided isolation procedure was used to separate the cytotoxic constituents from ethanolic extract of Dioscorea membranacea rhizomes by testing against five human cancer cell lines, i.e. large cell lung carcinoma, COR-L23; liver cancer cells, HepG2; prostate cancer cells, PC3; breast cancer cells MCF-7; cervical cancer cells, Hela; and one normal human lung cell line (MRC 5) using the SRB assay. Results: Two known dihydrophenanthrene compounds [2,4 dimethoxy-5,6-dihydroxy-9,10-dihydrophenanthrene (1) and 5-hydroxy-2,4,6-trimethoxy-9,10-dihydrophenanthrene (2)], and a new dihydrophenanthrene compound, 5,6,2 -trihydroxy 3,4-methoxy, 9,10-dihydrophenanthrene (3) were isolated and fully characterized. 1 showed the highest cytotoxic activity against COR-L23, MCF-7 and PC3 cell lines (IC50=14.89, 17.49 and 19.04 μM, respectively), and 2 showed selective cytotoxic activity against PC3 (IC50=23.54 μM). The new compound 3 showed selective cytotoxic activity against only MCF-7 cells (IC50=31.41 μM). Interestingly the crude extract of DM was much less toxic to the normal cell line (MRC-5) (IC50 > 50 μg/ml) compared to the five cancer cell lines, (IC50 value ranged between 6 and 29 μg/ml). Conclusion: The phytochemicals isolated from DM may serve as lead compounds for the design of new anticancer agents with better selective cytotoxic indices