Crude extracts of Witheringia solanacea leaves showed inhibition of NF-κB activation at 100 μg/mL induced by phorbol 12-myristate-13-acetate (PMA) in HeLa cells stably transfected with a luciferase reporter gene controlled by the IL-6 promoter. Three physalins were isolated from an active fraction, namely, physalins B (1), F (2), and D (3). Of these compounds, 1 and 2 demonstrated inhibitory activities on PMA-induced NF-κB activation at 16 and 8 μM and induced apoptosis after 24 h in a cell-cycle analysis using a human T cell leukemia Jurkat cell line. Compound 2 also inhibited TNF-α-induced NF-κB activation at 5 μM through the canonical pathway, but was inactive in the Tet-On-Luc assay, indicating specificity of action, although it interfered with Tet-On-Luc at higher concentrations. It is suggested that the presence of a double bond and an epoxy ring between carbons 5 and 6 in compounds 1 and 2, respectively (which are not present in compound 3), are related to their anti-inflammatory activity.