Purpose <p>Cashew nut testa (CNT) is an underutilized cashew by-product. This agro-industrial by-product has the potential for valorization. This study evaluates the novel potential of antioxidant, α-amylase, and α-glucosidase inhibitory activities of cashew nut testa polyphenols through bioassay-guided fractionation.</p> Methods <p>The antioxidant, α-glucosidase, and α-amylase enzyme inhibitory properties of crude and various phenolic fractions of cashew nut testa were evaluated in this study. Chromatographic separation and in-vitro bioassays directed the identification of the most active fraction.</p> Results <p>The crude ethanolic extract showed higher antioxidant activity from DPPH* (IC₅₀ 15.50 ± 0.67&#xa0;µg mL^− 1) and ABTS* (IC₅₀ 40.20 ± 2.40&#xa0;µg mL^− 1) assays. Among the column fractions, fraction 7 is the most potent fraction with superior antioxidant activity. Characterization was done by TLC, HPTLC, and HPLC, indicating the presence of flavonoids. Cashew nut testa phenolics showed potential inhibition towards α-amylase and α-glucosidase.</p> Conclusion <p>Polyphenols recovered from this agro-industrial by-product had antioxidant, α-amylase, and α-glucosidase inhibitory properties. Flavonoids, especially (+)-catechin, (-)-epicatechin, epigallocatechin, and catechin gallate, are the primary polyphenols responsible for the antioxidant, α-glucosidase and α-amylase inhibitory properties of cashew nut testa. This study suggests the potential valorization of this agro-industrial by-product as a source of antioxidants and a natural source of polyphenols to manage hyperglycaemia.</p>