Two new steroidal lactones of the withanolide‐type, 5β, 6α, 14α, 17β, 20β‐pentahydroxy‐1‐oxo‐20 S, 22R‐witha‐2, 24‐dienolide (1)and 6α, 7α‐epoxy‐5α, 14α, 17α, 23β‐tetrahydroxy‐1‐oxo‐22R‐witha‐2, 24‐dienolide (2), were isolated from the fruit of Withania somnifera (L.) Dunal (Solanaceae) growing in Southern Egypt, together with two known coumarins, scopoletin (3)and aesculetin (4), a known triterpene, viz. β‐amyrin (5), and two known phytosterols, viz. stigmasterol (6) and sitosterol (7). The structures of the isolated compounds have been elucidated by spectroscopic methods including UV, IR, 1H‐, 13C‐NMR, DEPT experiments, HRFAB‐, FAB‐, and EI‐mass spectrometry. The in vitro antimicrobial activity of the new withanolide (1) and the alcoholic fruit extract are discussed.