A new steroidal lactone of the Withanolide A series has been isolated from the supercritical fluid extract of Eucalyptus globulus L. (bark) as a major component (I) along with a known structurally similar steroidal lactone as minor component (II). The structural identification of the new lactone was accomplished by different spectroscopic techniques viz. 1 H and 13C NMR, etc. The relative stereochemistry was unequivocally determined from the X-ray crystallography.