Ethnopharmacological relevance: Eulophia nuda L. (Orchidaceae) is a medicinally important terrestrial orchid used for the treatment of tumours and various health problems by the local healers throughout the Western Ghats region in Maharashtra (India). Aim of the study: To isolate the active molecule from Eulophia nuda and to study its cytotoxic potential against human cancer cells. Materials and methods: The crude methanolic extract of Eulophia nuda tubers was fractionated by stepwise gradient of the solvents chloroform-methanol to isolate the pure compound. Isolated pure compound was assessed for its cytotoxic potential against human breast cancer cell lines, MCF-7 and MDA-MB-231 using MTT assay. Structure elucidation of the isolated active compound was carried out by extensive spectroscopic analysis including (1)H NMR, (13)C NMR, NOESY, COSY, LC-MS and IR. Results: The isolated active molecule was identified as phenanthrene derivative 9,10-dihydro-2,5-dimethoxyphenanthrene-1,7-diol. This compound showed good antiproliferative activity against human breast cancer cell lines MCF-7 (91%) and MDA-MB-231 (85%) at 1000 mu g/ml concentration. Conclusion: 9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol from Eulophia nuda tubers showed good growth suppressive effect against human cancer cell lines MCF-7 and MDA-MB-231 making it a potential biomolecule against human cancer. (C) 2009 Elsevier Ireland Ltd. All rights reserved.