Castanea sativa shells (CSS), an abundant and underexploited agro-residue in chestnut producing countries, are an excellent source of bioactive compounds with outstanding health-promoting properties. The aim of this study was to recover bioactive compounds from CSS employing Supercritical Fluid Extraction-CO2 (SC-CO2). The optimization of the extraction was performed based on the antioxidant activity of CSS extracts evaluated by FRAP, DPPH and ABTS assays. The optimal CSS extract was further characterized regarding in vitro radical scavenging capacity, phenolic profile and intestinal cell effects. The optimal conditions were 60 °C, 350 bar and 15% of co-solvent. The results demonstrated that CSS extract was an effective scavenger of NO (IC50 = 0.76 μg/mL) and HOCl (IC50 = 1.57 μg/mL). The IC50 calculated for Caco-2 and HT29-MTX were, respectively, 477.94 μg/mL and 3.71 μg/mL. Indeed, the phenolic profile revealed the presence of phenolic acids (particularly ellagic acid and caffeic acid derivative) and flavonoids (epigallocatechin and catechin/epicatechin). Overall, this work suggests the valorization of CSS extracted by SC-CO2 as promising nutraceutical ingredient based on its bioactivity and safety. To the best of our knowledge, this is the first study that employed SC to recover bioactive compounds from CSS.