The galls of Pistacia integerrima are used in folk medicine for curing diabetes. The main aim of this study was the purification of flavonoids from galls of P. integerrima. The methanolic extract was subjected to column chromatographic analysis which afforded six flavonoids, namely, 3,5,7,4 ′ -tetrahydroxy-flavanone (1), naringenin (2), 3,5,4 ′ -trihydroxy,7-methoxy-flavanone (3), sakuranetin (4), spinacetin (5), and patuletin (6). These isolated compounds (1–6) were tested against α-glycosidase. The maximum antagonistic effect was noted against compound 6 (97.65%) followed by compound 5 (90.42%) and compound 1 (90.01%) at the same concentration (0.2 μg). The inhibitory potential of all tested compounds was significant with a degree of variation from each other. Docking studies showed that all studied compounds interact with the active site residues via hydrogen bond interactions with hydroxyl groups, and thus, inhibition was enhanced. Hence, this finding would be the first screening of isolated flavonoids for α-glycosidase activity and with the mechanism of action. These flavonoids should be further investigated as candidate drugs for combating diabetes mellitus.