Gallotannins were extracted from mango (Mangifera indica L.) kernels with aqueous acetone (80%, v/v) and purified using liquid-liquid extraction and two-step low-pressure liquid chromatography (LPLC) on Sephadex LH-20. Analytical high-performance liquid chromatography and mass spectrometry confirmed the presence of hydrolyzable tannins with a degree of galloylation ranging from 4 to 9 and additionally revealed the presence of deca-, undeca-, and dodeca-O-galloylglucose. Further purification using two-step semipreparative HPLC resulted in three pure hydrolyzable tannins, penta-, hexa-, and hepta-O-galloylglucose, with antibacterial activity, as evidenced from the agar spot and critical dilution assays. Although the growth of lactic acid bacteria was not inhibited, the proliferation of Gram-positive food spoilage bacteria was prevented and the growth of Gram-negative Escherichia coli was reduced. Because bacterial growth could be restored by the addition of iron to the medium, this study strongly supports the view that the inhibitory effects of hydrolyzable tannins are due to their iron-complexing properties.