Abstract <p><i>Vitis vinifera</i> L. leaves represent one of the most phenolic-rich grapevine biomasses, exhibiting a broad spectrum of biological activities, including prebiotic and microbiota-modulating ones. However, as they are mainly regarded as agro-industrial waste, they are still an underexploited resource. Probiotic-driven fermentation may offer a promising strategy to enhance their bioactive potential while addressing this challenge. The aim of this study was to evaluate the effects of the in vitro fermentation of <i>Vitis vinifera</i> L. leaf extract (VVLE) by <i>Lactiplantibacillus plantarum</i> PBS067 in terms of phenolic content, antioxidant activity, lactic acid and protein content, and metabolomic profile. Our analysis shows the prebiotic activity of VVLE, as VVLE-supplemented medium allowed probiotic growth without toxic effects. Leaves fermentation led to a significant enrichment in lactic acid and the biotransformation of complex plant-secondary metabolites into smaller derivatives. However, VVLE antioxidant capacity slightly decreased after the fermentation, likely due to the phenolic conversion itself. These results highlight how controlled lacto-fermentation valorizes grapevine leaves, transforming them into sustainable upcycled bioactive products with potential applications within cosmetics, pharmaceuticals, nutraceuticals and complementary medical product sectors.</p> Key points <p>• <i>L. plantarum PBS067 thrives in medium supplemented with polyphenol-rich leaf extract</i></p> <p>• <i>L. plantarum PBS067-driven fermentation modulates VVLE phytochemical profile</i></p> <p>• <i>L. plantarum PBS067-driven fermentation valorizes viticulture by-products</i></p> Graphical abstract <p></p>