Flavonoid glycosides (FGs) appear to be good candidates for controlling blood glucose levels, so regular consumption of vegetables/fruits rich in FGs may prevent the consequences of type 2 diabetes (DM). Inhibition of digestive enzymes using natural FGs is a suitable dietary tool to regulate the hydrolysis of polysaccharides and overcome hyperglycemia. The aim of the current research is to find FGs that can effectively inhibit the digestive enzymes α-glucosidase (α-Gl) and α-amylase (α-Am). Accordingly, twenty-three FGs were selected and filtered through docking-based virtual screening. Based on the molecular docking and molecular dynamics (MD) simulation, among the 23 selected FGs, nicotiflorin and swertisin significantly inhibited α-Gl and α-Am, respectively. In vitro analysis revealed the inhibitory capacity of nicotiflorin on α-Gl was equal to IC50 at 0.148 mg/ml and the inhibitory activity of swertisin on α-Am was equal to IC50 at 1.894 mg/ml. It was found that nicotiflorin and swertisin act much like as a competitive inhibitor on α-Gl and α-Am, respectively. Furthermore, the fluorescence intensity of both enzymes decreased after interaction with two FGs. FT-IR and scanning electron microscopy (SEM) measurements suggested that the interactions could alter the conformation and microenvironment of the enzymes. Moreover, in vivo evaluation showed that the administration of nicotiflorin and swertisin can alleviate the blood glucose level of rats compared to the starch group (p < 0.05). The findings highlight that nicotiflorin and swertisin can be considered as possible inhibitors in treating diabetes mellitus via digestive enzymes inhibition.