Abstract: The tuber of Eulophia herbacea L. (Orchidaceae) is utilized as food particle with health benefit used by the tribes of Toranmal region, Maharashtra, India and also valued in management of cancer, in folklore medicine. The present study was undertaken to explore the cytotoxic potential of Eulophia herbacea L. tubers. The methanol extract and its fractions were tested using Brine Shrimp Lethality Test (BSLT) and human cancer cell line study. Furthermore the phytoconstituents was isolated from EA fraction and identified as 1-phenanthrenecarboxylic acid 1, 2, 3, 4, 4a, 9, 10, 10a-octahydro-1, 4a-dimethyl-, methyl ester (PCA). Mixtures of Phytosterols (MPS) and stigmasterol were obtained from n-hexane fraction. ME, EAF HEX, MPS and stigmasterol showed cytotoxic potential in brine shrimp lethality assay. PCA demonstrated most potent anticancer activity with GI50 concentration between 26.1 and 53.7 μg mL-1 for human cell line. HEX was active only against human Leukemia (K562) cell line with concentration <10 μg mL-1 of GI50. Overall, ethno pharmacological evaluations proved potency of activity in successive sequence of EAF>HEX>ME. The ethyl acetate fraction of methanol extract was found to be active in BSLT with LC50 168.3 μg mL-1, while the phenanthrene type of isolated compound from ethyl acetate fraction was showed as more active against a panel of three human cancer cells. TGI (Total Growth Inhibition) and GI50 of all samples were found to be less than 80 μg mL-1. Hence, the present study was confirmed the traditional anticancer effect of Eulophia herbacea tuber and could be a potential source for anticancer drugs.