We studied the defensive pygidial gland secretions in the adults of three ground beetle species of the genus Bembidion. We detected 46 chemical compounds, of which we successfully identified 37, while nine remained unidentified. There were 4, 45 and 42 compounds in the pygidial gland secretions of Bembidion (Peryphanes) dalmatinum, B. (P.) deletum, and B. (Peryphus) subcostatum, respectively. n-Undecane, methacrylic acid and tiglic acid were common in all the studied species. Methacrylic acid was most abundant in the secretions of B. (P.) dalmatinum and B. (P.) deletum, while isovaleric acid in the secretion of B. (P.) subcostatum. We identified 30 compounds new to the tribe Bembidiini, 12 of which were recorded for the first time in the family Carabidae. In addition, we also analyzed the morphology of the pygidial glands in B. (P.) dalmatinum using both bright-field and nonlinear microscopy.