Twelve bisdesmosidic cholestane glycosides (1-12), including nine new ones (1-9), were isolated from the bulbs of Ornithogalum thyrsoides by monitoring the cytotoxic activity on HL-60 leukemia cells. The structural assignment of the new compounds was carried out by spectroscopic analysis and the results of hydrolytic cleavage. The 3-O-monoglucosides with an aromatic acyl group at the C-16 diglycoside moiety (1, 12) were extremely cytotoxic, with respective IC(50) values of 0.00016 and 0.00013 microg/mL, and the other compounds, except for 2, 5, and 8, also showed cytotoxic activity as potent as etoposide (IC(50) 0.30 microg/mL), used as a positive control. These cholestanes were concluded to contribute to the potent cytotoxicity of the crude O. thyrsoides bulb extract.