In this study， Tween 80， sodium dodecyl sulfate (SDS) and cetylpyridine chloride (CPC) were used as emulsifiers to prepare thyme essential oil nanoemulsions (TEON) by ultrasonic nano-emulsification method. Moreover， the antibacterial effect and mechanism of TEON towards Escherichia coli O157:H7 were studied by analyzing the structure and function of bacterial cell membrane， together with ATPase activity and intracellular ROS. According to our results， three nanoemulsions showed significant differences among the alteration of function and structure of bacterial cell membrane， ATPase activity and ROS production. TEON-T80 significantly destroyed bacterial outer membrane permeability and fluidity， as well as inhibited the activity of ATPase； TEON-SDS showed excellent antibacterial effect， reducing E. coli by 8.17 lg (CFU/mL) within 40 minutes. In addition， TEON-SDS can also disturb the permeability of the bacterial outer and inner membrane， destroyed the outer membrane fluidity and ATPase activity； By contrast， the positively charged TEON-CPC significantly reduced the bacterial outer membrane fluidity and ATPase activity， generated a large amount of intracellular reactive oxygen species (ROS)， and destroyed the bacterial membrane structure. In conclusion， the antibacterial effect and mechanisms of three nanoemulsions were analyzed and compared， which provided a theoretical basis for the application of essential oil nanoemulsions in the control of foodborne pathogens.