The quest for eco-friendly antifungal compounds from natural sources has surged, seeking alternatives to synthetic fungicides. In this study, we explored Cupressus sempervirens organic extracts antifungal potential against Botrytis cinerea, a destructive fungus causing grey mold disease in crops. Extracts from various phenological stages were evaluated for their antifungal activities. The dichloromethanolic extract from the flowering stage exhibited the highest efficacy, completely inhibiting B. cinerea mycelial growth, at 250 μg/mL and preventing conidia germination at 500 μg/mL. Bioguided fractionation and chromatography, led to the identification of isoquercetin as the active compound responsible for the antifungal effects. These findings present promising possibilities for the development of sustainable biofungicides to combat grey mould disease in agriculture. Further investigations into isoquercetin's potential as a biofungicide are warranted.