In this study, two kinds of Aged garlic extract (AGE-1 and AGE-2) were prepared and the two samples were extracted by petroleum ether to obtain the petroleum ether fractions of AGE-1 and AGE-2. The minimum inhibitory concentration (MIC) of AGE-1 and AGE-2 against Escherichia coli was determined by broth microdilution method. The MIC of AGE-1 and AGE-2 against Escherichia coli was 31 μg/mL and 62 μg/mL, respectively. The antibacterial mechanism of AGE-1 and AGE-2 against Escherichia coli was studied by growth curves, leakage of cell contents, conductivity of culture medium, confocal and scanning electron microscope. Further research found that AGE-1 and AGE-2 inhibited the growth of Escherichia coli, damaged the cell wall and cell membrane, changed the permeability of cell membrane, which led to the leakage of electrolyte and macromolecules as well as cell apoptosis. The cell surface was rough and the cell was sticky and depressed.