The NLRP3 inflammasome is a complex present in cells of the innate immune system and involved in numerous inflammatory diseases, being a potential target for their treatment. NLRP3 inflammasome regulates caspase-1 activation and subsequent interleukin (IL)-1β and IL-18 release, and a type of cell death named pyroptosis. However, no specific NLRP3 inhibitors are clinically available to date. Dehydroisohispanolone (DIH) is a natural compound derived from the diterpene hispanolone with anti-inflammatory activity via inhibition of NF-κB activation. In this study, we evaluated whether DIH modulates NLRP3 inflammasome activation in macrophages. Our findings revealed that DIH inhibited NLRP3 activation in J774A.1 macrophages triggered by diverse stimuli (LPS plus nigericin/ adenosine triphosphate/ monosodium urate crystals), as it reduced IL-1β release and caspase-1 activation. DIH treatment also diminished cleaved IL-1β and caspase-1 p10 expression, although expression of NLRP3, ASC, pro-IL-1β and pro-caspase-1 was not affected. Pyroptosis mediated by NLRP3 activation was also attenuated by DIH. In addition, we found that DIH acts as a dual NLRP3 inhibitor by inhibition of LPS-induced priming step in NLRP3 inflammasome activation. Similar results on IL-1β release were observed in nigericin-activated bone marrow-derived macrophages. Covalent molecular docking study of DIH onto the ATP-binding site revealed that DIH binds to NLRP3, forming a covalent bond with Cys415. In conclusion, our experiments show that DIH is an effective NLRP3 inflammasome inhibitor, by inhibiting both the priming and the activation steps, making DIH a promising therapeutic agent for the treatment of inflammatory-related diseases.