Methanolic and ethanolic crude extracts of Vitis vinifera canes exhibited significant antifungal activity against the three major fungal pathogens affecting grapevines, Plasmopara viticola, Erysiphe necator and Botrytis cinerea. The active extracts were analyzed by LC-PDA–ESI-MS, and selected compounds were identified. Efficient targeted isolation using medium-pressure liquid chromatography afforded six pure constituents in one step. The structures of the isolated compounds were elucidated by NMR and HRMS. Six identified compounds (ampelopsin A, hopeaphenol, trans-resveratrol, ampelopsin H, ε-viniferin, and E-vitisin B) presented antifungal activities against P. viticola. ε-Viniferin also exhibited a low antifungal activity against B. cinerea. None of the identified compounds inhibited the germination of E. necator. The potential to develop a novel natural fungicide against the three major fungal pathogens affecting V. vinifera from viticulture waste material is discussed.