Monolaurin is a nontraditional antimicrobial agent that possesses better antimicrobial activities but causes no health problems to consumers, but the use of monolaurin in the food industry as a preservative is still limited. Using a microtiter plate assay, the minimum inhibitory concentrations for monolaurin were 25 microg/mL against Escherichia coli, 12.5 microg/mL against Staphylococcus aureus, and 30 microg/mL against Bacillus subtilis. The interaction with commonly used antimicrobials revealed that monolaurin and nisin acted synergistically against the test microorganisms, monolaurin in combination with sodium dehydroacetate or ethylenediaminetetraacetic acid was synergistic against E. coli and B. subtilis but not S. aureus, and monolaurin combined with calcium propionate or sodium lactate showed no synergistic effects against any test microorganism. The interaction with food components revealed that the antibacterial effectiveness of monolaurin was reduced by fat or starch while the monolaurin activity remained unchanged in the presence of protein. This study contributes to a better understanding on the use of monolaurin as a nontraditional preservative in food products. Results from this study suggest the potential use of monolaurin as a nontraditional preservative in combination with commonly used antimicrobials, such as nisin, sodium dehydroacetate, or ethylenediaminetetraacetic acid, and suggest that the antibacterial effectiveness of monolaurin may be reduced significantly in high-fat or low-starch food products.