Abstract: The aim of this work was to explore the antimicrobial activity and mechanism of monoglycerides containing monolaurin and monocaprin. Paper Disc Method was used to evaluate bacteria susceptibility of monolaurin and monocaprin. Two-Fold Dilution Method was used to test minimum inhibitory concentration (MIC), and Photoelectricity Turbidimetry Method was used to test antibacterial efficacy. Orescein diacetate (FDA) was used as fluorescent indicator to reflect the membrance permeability of testing bacteria after treating with monolaurin and monocaprin. Spectropbotometry Method was used to test the leak of internal substance of testing bacteria, such as: protein, nucleic acid, lipopolysaccharide, K+, Ca2+ and Mg2+. The change of cell surface was characterized using scanning electron microscope. Result showed that gram-positive and gram-positive bacteria were susceptive to monolaurin and monocaprin. Activity of monolaurin and monocaprin against Bacillus subtilis (G+) and Staphylococus aureus (G+) resulted from the destruction of cell wall and membrance caused by monolaurin and monocaprin. On the contrary, its antibacterial activity to Escherichia coil (G-) resulted from the leaking of lipopolysaccharide in the cell wall.