Eight undescribed and two known withanolides were obtained from the aerial parts of Physalis minima. The structures of these compounds were defined by spectroscopic methods including 1D and 2D NMR, HRESIMS, and electronic circular dichroism (ECD) data analysis. Physminin E was elucidated to be a rare 13,14-seco-withanolide. Inhibitory effects of these compounds on nitric oxide (NO) production were evaluated by using LPS-activated RAW264.7 macrophages, and physminin C was shown to be the most active with an IC50 value of 3.5 μM. The further mechanistic analysis of NO inhibition was performed by molecular docking and Western blotting.