Orchids of the genus Bulbophyllum have been reported to possess antitumor activity. Present study investigated the possible antitumor activity of the active fraction of bulb and root of Bulbophyllum sterile. Alcoholic extract along with petroleum ether, dichloromethane and ethyl acetate fractions were subjected to SRB assay in HCT-116, MDA-MB-231 and A549 cell lines. The active fractions were further evaluated for apoptosis, expression of apoptotic signaling proteins, comet assay and cell cycle analysis. Furthermore, they were assessed for in vivo antitumor activity in Ehrlich ascites carcinoma model. Petroleum fraction of bulbs (PFB) and roots (PFR) was found to be most active in HCT-116 cell lines with IC₅₀ value of 94.2±6.0 and 75.7±9.8, respectively. Apoptosis was evident from acridine orange/ethidium bromide staining along with the expression of phospho-p53 and phospho-Bad. Both PFB and PFR arrested G₂/M phase of the cell cycle with 32.6% and 49.4% arrest, respectively compared to 17.5% arrest with control. An increase in mean life span and hepatic antioxidant levels was observed with PFB and PFR treatment in EAC inoculated mice. The results suggested that the active fractions of bulbs and roots possess anticancer activity likely by inducing apoptosis through phospho-p53 dependent pathway.