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Microbial pathogens e.g., S. aureus can survive and spread in food cold chain. Under low temperature, microbes develop resistance to common antibacterial agents. Thus, it is important to identify alternative strategies to control pathogens in food cold chain. In present study, we discovered that thymol, different from other tested essential oils, displayed improved ability to kill S. aureus under frozen condition (−20 °C). Time-killing kinetics showed that 99.99% S. aureus were killed after exposure to 100 μg/mL thymol within 6 h, and such bactericidal activity was maintained even after multiple cycles of freezing and thawing. In contrast to the ROS-dependent bactericidal mechanism at 37 °C, thymol killed S. aureus in a ROS-independent mode at −20 °C. The proteomic analysis supported that thymol killed S. aureus by directly destroyed the stability of cell membrane without intracellular biological responses. Under frozen condition, thymol efficiently prevented bacteria contamination in low-lipid foods without affecting the foods' hardness, chewiness, resilience, adhesiveness, springiness and cohesiveness. These findings uncovered a new mechanism of thymol-mediated bactericidal activity under frozen condition and suggested an alternative efficient strategy to control bacterial contamination in cold chain food. • Identified the best bactericidal ability of thymol at −20 °C. • Revealed the mechanism of thymol to eradicate S. aureus in a ROS-independent manner under low temperature. • Provided new insights into the strategy to combat S. aureus contamination in cold chain food. [ABSTRACT FROM AUTHOR]