Three new withanolides, designated as withatatulins B, C, and D, were isolated from the fresh flowers of Datura tatula Linn. Detailed spectral analysis of these compounds permitted advancement of their structures respectively, as $5{eta},6{eta}-epoxy-12{eta}$,21-dihydroxy-1-oxo-witha-2,24-dienolide (2), $6{eta},12{eta},21-trihydroxy-1-oxowitha$ 2, 4,24-trienolide (3) and $5{eta},6{eta},12{eta}$,21-tetrahydroxy-1-oxo-witha-2,24-dienolide (4a). Withanolides with oxygen functions both at 12 and 21-positions are rare and first reported from Datura species.