Sixteen new withanolides, physangulatins A-N (1-14) and withaphysalins Y and Z (15 and 16), as well as 12 known analogues, were isolated from the stems and leaves of Physalis angulata L. Their structures were established using extensive spectroscopic data analyses. The absolute configurations of 1 and 9 were assigned via X-ray crystallography. The isolated compounds were tested for their antiproliferative effects against human prostate cancer cells (C4-2B and 22Rvl), human renal carcinoma cells (786-O, A-498, and ACHN), and human melanoma cells (A375-S2), as well as inhibitory effects on NO production induced by LPS in macrophages. Compounds 9, 17, 20, 21, 25, and 27 showed antiproliferative effects against all tested cancer cells, with IC50 values of 0.18-7.43 μM. Compounds 3-5, 9-11, 17, 20-22, 24, 25, and 27 displayed inhibitory effects against NO production, with IC50 values of 1.36-11.59 μM.