Diabetes is a major health problem that is associated with high risk of various complications. Medicinal plants hold great promise against diabetes. The traditional use of Cleome droserifolia as an antidiabetic agent was correlated to its flavonol glycosides content. In the current study, five major flavonol glycosides appeared on the RP-HPLC chromatogram of the aqueous extract namely; quercetin-3-O-β-d-glucosyl-7-O-α-rhamnoside (1), isorhamnetin-7-O-β-neohesperidoside (2), isorhamnetin-3-O-β-d-glucoside (3) kaempferol-4′-methoxy-3,7-O-α-dirhamnoside (4), and isorhamnetin-3-O-α-(4″-acetylrhamnoside)-7-O-α-rhamnoside (5). The inhibitory activities of these compounds were tested in vitro against several enzymes involved in diabetes management. Only the relatively less polar methoxylated flavonol glycosides (4, 5) showed mild to moderate α-amylase and α-glucosidase inhibitory activities. Compounds 1–4 displayed remarkable inhibition of dipeptidyl peptidase IV (DPPIV) enzyme (IC50 0.194 ± 0.06, 0.573 ± 0.03, 0.345 ± 0.02 and 0.281 ± 0.05 µg/mL, respectively) comparable to vildagliptin (IC50 0.154 ± 0.02 µg/mL). Moreover, these compounds showed high potential in preventing diabetes complications through inhibiting aldose reductase enzyme and combating oxidative stress. Both isorhamnetin glycoside derivatives (2, 3) exhibited the highest activities in aldose reductase inhibition and compound 2 (IC50 5.45 ± 0.26 µg/mL) was even more potent than standard quercetin (IC50 7.77 ± 0.43 µg/mL). Additionally, these flavonols exerted excellent antioxidant capacities through 2, 2-diphenyl-1-picrylhydrazil (DPPH) and ferric reducing antioxidant (FRAP) assays.