The antibacterial effect of Litsea cubeba oil on wild strains of Cronobacter sakazakii in infant formula milk powder is currently unknown. Thus, the continuous double dilution, plate counting, and growth curve methods were used to investigate the antibacterial mechanism by detecting the β-galactosidase activity, alkaline phosphatase activity, intracellular DNA content, and bacterial structure changes. The results showed a strong antibacterial effect for L. cubeba oil against C. sakazakii, with a minimum inhibitory concentration (MIC) of 6.62 μg•mL-1 and significant inhibition in the growth of C. sakazakii after one treatment. The activity of extracellular alkaline phosphatase and intracellular β-galactosidase in C. sakazakii showed significant 2- and 13.50-fold increases as compared to the control (P<0.05), and the cell morphology of the bacteria was destroyed. The cell contents were leaked, with the leaked nucleic acid and protein reaching 1.03 times that observed in the control (P<0.05). The intracellular DNA content was reduced to 0.17 μg•mL-1, the DAPI fluorescence area was decreased, the brightness of the gel block electrophoresis bands darkened, and the DNA was damaged. In summary, L. cubeba essential oil could be utilized as a highly effective bacteriostatic agent for C. sakazakii due to its action in destroying both the cell membrane and the DNA and interfering with the normal physiological activity of the bacteria, resulting in death. This study is expected to provide a theoretical basis for the development of the essential oil of L. cubeba as a new antibacterial agent.