Botrytis cinerea and Plasmopara viticola, the causal agents of grey mold and downy mildew, respectively, are two major grapevine pathogens whose control largely relies on synthetic fungicides, raising environmental and health concerns. Plant-derived secondary metabolites, particularly flavonoids involved in plant defense, represent promising sustainable alternatives. Among them, sakuranetin, a flavanone aglycone known for its antifungal activity in rice, remains poorly explored for grapevine protection. In this study, sakuranetin was purified from cherry branches (48 mg) and structurally characterized using UHPLC-ESI-QTOF-MS and NMR analyses. Its antifungal activity against B. cinerea and P. viticola was evaluated through in vitro, in vivo and in planta assays. For B. cinerea, our results showed a significant in vitro inhibition of mycelium growth, with EC₅₀ values of 16.43 mg·L⁻¹, while no protection of detached berries was observed. Against P. viticola, sakuranetin has no effect on the release of zoospores, but there is a total inhibition of spore germination at 1 mg·L⁻¹ in vitro, confirmed in vivo on a foliar disc. In planta, no significant protection is observed at 25 mg·L⁻¹, even if some targeted defense genes are induced. Further studies are needed to determine the best concentration of sakuranetin to use to manage B. cinerea and P. viticola in planta.