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Lignocellulosic biomasses have the potential to generate by-products with biological activity (i. e., polyphenols) as well as biopolymers (i. e., cellulose, hemicellulose, pectins, lignin). The wine industry is one of the pillars of Italian agri-food sector. Nevertheless, large quantities of by-products such as grape stems are produced, which are usually disposed of at a cost, and therefore represent an attractive negative-cost feedstock for biorefinery. In this work, a sequential protocol for biomass valorization is proposed, characterized by a multidisciplinary strategy using enabling technologies and subcritical water as a green solvent, where physical/chemical treatments synergistically interact with biological treatments. The first phase involved the sequential fractionation of grape stalks, obtaining several product streams rich in polyphenols, hemicellulose, pectin (13.15 % of cumulative yield on biomass), lignin and cellulose. A membrane treatment was employed to recycle materials within the process. Finally, the cellulose-rich residue was exploited as a fermentation substrate for the last step, producing up to 5.8 g/L of lactic acid by harnessing suitably engineered Clostridium thermocellum strains. The polyphenolic fraction successfully inhibited the growth of Brettanomyces bruxellensis and Acetobacter pasteurianus, microorganisms responsible for major wine off-flavors. Globally, this study represents a proof-of-concept of a second-generation biorefining process based on locally available waste biomass., (© 2025 The Author(s). ChemSusChem published by Wiley-VCH GmbH.)