A chlorinated withanolide, 6α-chloro-5β,17α-dihydroxywithaferin A (1), and nine known withanolides, 6α-chloro-5β-hydroxywithaferin A (2), (22R)-5β-formyl-6β,27-dihydroxy-1-oxo-4-norwith-24-enolide, withaferin A, 2,3-dihydrowithaferin A, 3-methoxy-2,3-dihydrowithaferin A, 2,3-didehydrosomnifericin, withanone, withanoside IV and withanoside X, were isolated from Withania somnifera (Solanaceae). All structures were elucidated on the basis of spectroscopic methods (IR, HRESIMS, 1D/2D NMR). X-ray crystallography confirmed the absolute configuration of 1.Graphical abstractTen withanolides including one previously unknown chlorinated compound (1) were isolated from Withania somnifera (Solanaceae). Spectroscopic methods and X-ray crystallography elucidated the absolute configuration of 1.Highlights► From Withania somnifera, one new chlorinated withanolide, 6α-chloro-5β,17α-dihydroxywithaferin A (1), was isolated together with nine known ones. ► Full NMR assignments for two chlorinated withanolides (1) and (2) were described. ► X-ray crystallography confirmed the absolute configuration of 1.