Ten new withanolides, including four perulactone-type withanolides, perulactones E−H (1–4), three 28-hydroxy-withanolides, withaperuvins I−K (5–7), and three other withanolides, withaperuvins L−N (8–10), together with six known compounds (11–16) were isolated from the aerial parts of Physalis peruviana. The structures of these compounds were elucidated on the basis of extensive spectroscopic analyses (1D and 2D NMR, IR, HR-MS) and chemical methods.Highlights► Sixteen withanolides were isolated from the aerial parts of Physalis peruviana. ► Their structures were determined by MS and NMR analysis, as well as chemical methods. ► Perulactones E–H (1–4) belong to the small group of perulactone-type withanolides.