The phenomenon of alpha-glucosidase inhibition has attracted the attention of researchers due to its association with type 2 diabetes treatment in humans. In this study, we found that 2-thiobarbituric acid (TBA) induces complex inhibition of alpha-glucosidase using kinetics tests and molecular dynamics (MD) simulations. Computational MD and docking simulations demonstrate that TBA interacts with three residues on active sites of alpha-glucosidase such as Met69, Arg212, and His348. These biochemical tests indicate that TBA reversibly inhibits alpha-glucosidase in a parabolic noncompetitive manner (IC50 = 17.13 +/- 1.14 mM; K-i = 13.25 +/- 0.56 mM) and that this inhibition is accompanied by a biphasic kinetic process. The tertiary conformational changes were not synchronized with TBA inhibition but we observed hydrophobic disruption after inactivation at higher concentrations of TBA. Our results provide insight into the functional roles of residues located at the active sites of alpha-glucosidase, and we suggest that compounds similar to TBA (heterocyclic compounds) targeting the key residues of active sites are potential alpha-glucosidase inhibitors.