Abstract BACKGROUND Gray mold, caused by Botrytis cinerea , is a widespread plant disease characterized by a broad host range and being difficult to control, leading to substantial economic losses. The extensive use of conventional chemical fungicides has raised environmental concerns and induced resistance in B. cinerea , highlighting the urgent need for eco‐friendly, low‐toxicity alternatives. RESULTS This study evaluated the antifungal activity of xanthohumol, a secondary metabolite derived from hops, against B. cinerea and investigated its mechanism of action. The results showed that xanthohumol exhibited inhibitory effects against multiple plant pathogenic fungi. It significantly suppressed both mycelial growth and spore germination of B. cinerea , with EC 50 values of 17.37 and 49.68 μg/mL, respectively. Furthermore, xanthohumol exhibited significant efficacy in controlling gray mold on tomato fruits and leaves. Scanning electron microscopy observations revealed that xanthohumol induced shrinkage, wrinkling, and collapse of hyphae. Physiological and biochemical analyses indicated that it disrupted fungal cell membrane integrity and permeability, enhanced antioxidant enzyme activities, induced reactive oxygen species (ROS) accumulation, and disturbed intracellular redox homeostasis. Integrated transcriptomic and metabolomic analyses, combined with molecular docking simulations, suggest that the antifungal activity of xanthohumol may primarily stem from the impairment of fungal pathogenicity and disruption of cellular structural integrity. Conclusion As a natural product, xanthohumol shows strong potential for development as a botanical fungicide and represents a promising candidate for the eco‐friendly control of gray mold. © 2026 Society of Chemical Industry.