This study optimized the ultrasound emulsification process using response surface methodology (RSM) to obtain thyme essential oil nanoemulsions (TEON) with excellent dispersion characteristics and antibacterial activity. Based on the single-factor experiments, the effects of ultrasonic power (350–550 W), time (5–15 min) and emulsifier [Tween 80 (T80), sodium dodecyl sulfate (SDS), and cetylpyridinium chloride (CPC)] concentrations were determined on response variables, including droplet size, PDI, zeta-potential, MIC, and MBC. Further analysis of stability and antibacterial mechanism were also conducted based on the optimal results. TEON-T80, obtained at 527.45 W, 9.97 min, and 5.94 mg/mL with good stability, killed E. coli through extracellular release, whereas TEON-CPC obtained at 350 W, 14.44 min, and 0.15 mg/mL exhibited excellent antibacterial activity through electrostatic interaction. TEON-SDS obtained at 350 W, 15 min, and 1 mg/mL was poor in stability and antibacterial activity. All nanoemulsions destroyed cell morphology and caused severe cytoplasm leakage. Through a preliminary antibacterial mechanism study, we hypothesized a controlled in vitro release for TEON-T80, electrostatic interaction for TEON-CPC, and self-assembly together with cell penetration for TEON-SDS. Considering all the results, TEON-CPC was determined as the best nanoemulsion for optimal antibacterial activity. Also, optimizing the ultrasonication process is useful for preparing TEON with enhanced dispersion characteristics and antibacterial activity. • Optimizing ultrasonication induced thyme essential oil nanoemulsions (TEON). • Tween 80, SDS, and cetylpyridinium chloride (CPC) were selected as emulsifiers. • Optimized TEON with Tween 80 exhibited excellent storage stability. • Optimized TEON with CPC showed the strongest antibacterial activity on E. coli. • TEON-CPC showed bactericidal effect through strong electrostatic interaction. [ABSTRACT FROM AUTHOR]