| Summary: | <i>Litsea cubeba</i> essential oil (LC-EO) has anti-insecticidal, antioxidant, and anticancer proper-ties; however, its antimicrobial activity toward <i>Cronobacter sakazakii</i> has not yet been researched extensively. The objective of this study was to investigate the antimicrobial and antibiofilm effects of LC-EO toward <i>C. sakazakii</i>, along with the underlying mechanisms. The minimum inhibitory concentrations of LC-EO toward eight different <i>C. sakazakii</i> strains ranged from 1.5 to 4.0 μL/mL, and LC-EO exposure showed a longer lag phase and lower specific growth compared to untreated bacteria. LC-EO increased reactive oxygen species production, decreased the integrity of the cell membrane, caused cell membrane depolarization, and decreased the ATP concentration in the cell, showing that LC-EO caused cellular damage associated with membrane permeability. LC-EO induced morphological changes in the cells. LC-EO inhibited <i>C. sakazakii</i> in reconstituted infant milk formula at 50 °C, and showed effective inactivation of <i>C. sakazakii</i> biofilms on stainless steel surfaces. Confocal laser scanning and attenuated total reflection–Fourier-transform infrared spectrometry indicated that the biofilms were disrupted by LC-EO. These findings suggest a potential for applying LC-EO in the prevention and control of <i>C. sakazakii</i> in the dairy industry as a natural antimicrobial and antibiofilm agent.
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