One of the most prevalent chronic metabolic disorders, diabetes mellitus, is defined by chronic hyperglycemia and the emergence of microvascular and macrovascular pathology unique to diabetes. Several diabetes problems are primarily caused by persistent hyperglycemia. In this study, aldose reductase (AR) and sorbitol dehydrogenase (SDH) enzymes were purified using chromatographic techniques from sheep kidneys and α-glycosidase purchased commercially. The inhibitory effect of seven acylthiourea compounds on the activity of these enzymes associated with diabetes was investigated. As a result of the studies, the acyl thioureas showed an inhibition effect at the micromolar level on the activity of studied enzymes. The most active compound dimethyl 1-((4-methoxybenzoyl)carbamothioyl)-5,5-diphenylpyrrolidine-2,4-dicarboxylate (2 c) had an excellent selectivity on AR with K i : 0.200 � 0.024 μM and dimethyl 1-((2,4-dichlorobenzoyl)carbamothioyl)-5,5-diphenylpyrrolidine-2,4-dicarboxylate (2 g) on SDH and αglycosidase with K i : 0.114 � 0.01 μM and K i : 055 � 0.01 μM, respectively. The findings suggest that our study will contribute to the development of innovative and/or alternative treatment strategies in the treatment of diabetes in the future since acylthiourea compounds have an effective inhibition capacity on critical enzymes associated with diabetes.