Five new withanolides, designated as 15-oxo-nicaphysalin B, 6β,7α-dihydroxynicandrenone 10, 24α,25β-dihydroxy-nicandrenone-2 and a mixture of the epimers 17-(1α/1β-methylpropanone)nicandrenone, in addition to six known others, were isolated from the leaf acetone extract from Nicandra physalodes.The complete structures of the five new withanolides, particularly their relative stereochemistries, were established by extensive spectroscopic analyses, including 1D and 2D nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HRESIMS).The main isolated withanolides were evaluated for their antibacterial, antifungal and larvicidal properties, but, except nicandrenone, which showed marginal larvicidal activity, none of them was active.