This study aimed to evaluate the antioxidant activity, the inhibitory effect on digestive enzymes linked to diabetes and the phytochemical composition of an active sub-extract obtained from Cousinia aintabensis (Asteraceae). The antioxidant effect of extracts was investigated with DPPH●, ABTS●+ and FRAP tests and the inhibition effect was tested on α-amylase and α-glucosidase. The quantitative and qualitative determination of the compounds within the active extract was carried out by LC-MS/MS. The highest total phenolic (185.816 ± 0.003 mg GAE/g extract) and total flavonoid content (147.423 ± 0.003 mg CA/g extract) was found in ethyl acetate sub-extract of C. aintabensis (CAE). It also showed the highest antioxidant activity on DPPH● (IC50 = 0.473 ± 0.056 mg/mL), ABTS●+ (at a conc. of 0.5 mg/mL equivalent to 0.543 ± 0.005 µM Trolox) and FRAP (at a conc. of 1 mg/mL equivalent to 2586 ± 6.421 mmol Fe2+) tests. Furthermore, the highest α-amylase and α-glucosidase activity were also found in CAE (IC50 = 0.361 ± 0.010 µg/mL for α-glucosidase and 1.010 ± 0.010 for α-amylase). According to LC-MS/MS analysis, the main compounds of CAE were cynarin and isorhamnetin 3-O-rutinoside.