AimsThe antimicrobial activity of Acacia farnesiana against Vibrio cholerae has been demonstrated; however, no information regarding its active compound or its mechanism of action has been documented. Methods and ResultsThe active compound was isolated from A.farnesiana by bioassay-guided fractionation and identified as methyl gallate by nuclear magnetic resonance (NMR) techniques (H-1 NMR and C-13 NMR). The minimum bactericidal concentration (MBC) of methyl gallate and its effect on membrane integrity, cytoplasmic pH, membrane potential, ATP synthesis and gene expression of cholera toxin (ctx) from V.cholerae were determined. The MBC of methyl gallate ranged from 301 to 501gml(-1). Methyl gallate affected cell membrane integrity, causing a decrease in cytoplasmic pH (pH(in), from 73 to <30), and membrane hyperpolarization, and ATP was no longer produced by the treated cells. However, methyl gallate did not affect ctx gene expression. ConclusionsMethyl gallate is a major antimicrobial compound from A.farnesiana that disturbs the membrane activity of V.cholerae. Significance and Impact of the StudyThe effects of methyl gallate validate several traditional antimicrobial uses of A.farnesiana, and it is an attractive alternative to control V.cholerae.