P2X7 receptor promotes inflammatory response and neuropathic pain. New drugs capable of impairing inflammation and pain-reducing adverse effects extracted from plant extracts have been studied. Physalis angulate L. possesses traditional uses and exhibits antiparasitic, anti-inflammatory, antimicrobial, antinociceptive, antimalarial, antileishmanial, immunosuppressive, antiasthmatic. diuretic, and antitumor activities. The most representative phytochemical constituents identified with medicinal importance are the physalins and withanolides. However, the mechanism of anti-inflammatory action is scarce. Although some physalins and withanolides subtypes have anti-inflammatory activity, only four physalins subtypes (B, D, F, and G) have further studies. Therefore, we evaluated the crude ethanolic extract enriched with physalins B, D, F, and G from P. angulata leaves, a pool containing the physalins B, D, F, G, and the physalins individually, as P2X7 receptor antagonists. For this purpose, we evaluated ATP-induced dye uptake, macroscopic currents, and interleukin 1-β (IL-1β) in vitro. The crude extract and pool dose-dependently inhibited P2X7 receptor function. Thus, physalin B, D, F, and G individually evaluated for 5′-triphosphate (ATP)-induced dye uptake assay, whole-cell patch-clamp, and cytokine release showed distinct antagonist levels. Physalin D displayed higher potency and efficacy than physalin B, F, and G for all these parameters. In vivo mice model as ATP-induced paw edema was potently inhibited for physalin D, in contrast to physalin B, F, and G. ATP and lipopolysaccharide (LPS)-induced pleurisy in mice were reversed for physalin D treatment. Molecular modeling and computational simulation predicted the intermolecular interactions between the P2X7 receptor and physalin derivatives. In silico results indicated physalin D and F as a potent allosteric P2X7 receptor antagonist. These data confirm physalin D as a promisor source for developing a new P2X7 receptor antagonist with anti-inflammatory action.