Within the framework of agro-industrial waste valorisation, this work reports the extraction and biological evaluation of bioactive compounds obtained from pruning residues of Vitis vinifera and Olea europaea. Microwave-assisted extraction was applied to different biomass fractions to obtain extracts subsequently assessed for antioxidant and anticancer activities using chemical and cell-based assays. Extracts derived from Olea europaea pruning residues exhibited strong antioxidant properties even without prior separation of leaves and branches, highlighting a simplified and efficient valorisation strategy. In parallel, Vitis vinifera pruning wood extracts showed significant antitumoral activity against glioblastoma and breast cancer models, with insights into their mechanisms of action. Furthermore, enantiopure forms of (E)-ε-viniferin were isolated and biologically investigated, demonstrating that chirality plays a key role in modulating anticancer activity and cytotoxic mechanisms. Overall, these results underline the high potential of pruning residues as sustainable sources of high-value bioactive molecules. This document is a deliverable of the project VINO ("VIneyard management for viNeprOduction"), which proposes research for sustainable innovation supported by the digitalisation of the wine sector with the primary objective of creating new solutions and readapting existing practices to cope with the effects of climate change in viticulture. This document is part of the project NODES which has received funding from the MUR – Missione 4, Componente 2, Investimento 1.5 – Creazione e rafforzamento di “Ecosistemi dell’innovazione”, costruzione di “leader territoriali di R&S'' – del PNRR funded by the European Union - NextGenerationEU with grant agreement no. ECS00000036