Biomass represents a natural source of valuable components with potential application in many industries. Recently, it has been demonstrated that vineyard pruning residues can be used as a potential source of bioactive compounds, namely polyphenols [1,2]. In this study, an experimental design approach was used to optimize the total phenolic content and antioxidant activity of vine-canes from Touriga Nacional variety. Subcritical water extraction (SWE) was employed, and the independent parameters studied were extraction temperature (150-280 °C) and time (20-50 min). A central composite design was used to evaluate the influence of extraction parameters in achieving higher recovery of phenolics, as well as higher antioxidant activities (by FRAP and ABTS assays). The optimal extraction conditions were 33 min and 280 °C, revealing a high total phenolic content (229 ± 23 mg gallic acid equivalents/g dw), as well as a high antioxidant activity by the FRAP and the ABTS assays (228 ± 20 and 236 ± 11 mg ascorbic acid equivalents/g dw, respectively). The extract also displayed strong in vitro neuroprotective effects, inhibiting several brain enzymes involved in neurodegeneration: acetylcholinesterase (IC50 = 290.5 µg/mL), butyrylcholinesterase (IC50 = 244.0 µg/mL), tyrosinase (IC50 = 1459.0 µg/mL) and monoamine oxidase B (~53.4 % of inhibition at 1000 µg/mL). The phenolic composition determined by HPLC-DAD revealed the presence of compounds belonging to different families, with gallic acid (118 ± 6 mg/100 g dw), catechin (468 ± 23 mg/100 g dw) and quercetin (153 ± 8 mg/100 g dw) being the major contributors to the demonstrated antioxidant properties of the produced vine-cane extracts. The extracts were evaluated regarding the presence of 14 organochlorine pesticides and the absence of these compounds was confirmed. The results proved that SWE can be a useful extraction technique for obtaining phenolic compounds from vineyard pruning residues, which can be further safely applied to food or cosmetic industries creating an added value to this residue. Acknowledgements: This research was funded by FCT/MCTES through national funds (UIDB/50006/2020 and UIDP/50006/2020.). This work was also financed by the FEDER Funds through the Operational Competitiveness Factors Program—COMPETE and by National Funds through FCT within the scope of the project “PTDC/BII-BIO/30884/2017—POCI-01-0145-FEDER-030884”. OD is thankful for the research grant from project PTDC/BII-BIO/30884/2017—POCI-01-0145-FEDER-030884. MMM (CEECIND/02702/2017), EFV (CEECIND/03988/2018), VCF (SFRH/BPD/109153/2015), FR (CEECIND/01886/2020), CG (CEECIND/03436/2020) and AFP (CEECIND/01614/2020) are grateful for the financial support financed by national funds through FCT—Fundação para a Ciência e a Tecnologia, I.P. and to REQUIMTE/LAQV. The supply of the vineyard pruning is acknowledged to Sogrape, S.A. [1] Moreira MM, Rodrigues F, Dorosh O, Pinto D, Costa PC, Švarc-Gajić J,Delerue-Matos C. Molecules, 2020, 25, 2969. [2] Dorosh O, Moreira MM, Pinto D, Peixoto AF, Freire C, Costa P, Rodrigues F, Delerue-Matos C. Foods, 2020, 9, 872.