Microemulsions are colloidal nanodispersions of oil and water stabilized by an interfacial film of surfactant molecules, typically in conjunction with a cosurfactant. There is a limited number of reports in the literature on microemulsion use for antimicrobial purposes. The physicochemical characterization of a food-grade fully dilutable microemulsion system with glycerol monolaurate (GML) as oil, organic acids as cosurfactant, Tween 80 as surfactant, and the antimicrobial activities against Escherichia coli and Staphylococcus aureus have been studied in this paper. The influence of organic acids on oil solubilization was clearly reflected in the phase behavior of these systems. Propionic acid demonstrated the greatest capability to improve the oil solubilization among the tested linear and nonlinear chain organic acids and contributed to the formation of U-type microemulsion systems. One microemulsion formulation with an average particle size of 8nm was selected, the composition is GML/propionic acid/Tween 80/water=3:9:8:12. The kinetics of killing experiments demonstrated that the undiluted microemulsion caused a complete loss of viability of E. coli or S. aureus cells in 1min and still had effective bactericidal effects even when diluted, more than 99% viable E. coli cells were killed within 15min and a complete loss of viability was achieved at 45min while more than 99% viable S. aureus cells were killed within 30min and a complete loss of viability was achieved at 60min in the presence of the 10-fold diluted microemulsion. The fast killing kinetics of the ten-fold serial dilutions of microemulsions were in good agreement with the mode of action studies, indicating that the interaction between the antimicrobial microemulsions and bacterial membranes significantly decreased the bacterial cell surface hydrophobicity and induced the quick release of 260nm absorbing materials. This work suggests the potential use of food-grade fully dilutable microemulsions for antimicrobial purposes in beverages or seafood products.