Eugenol is a versatile plant essential oil, but its high volatility and low water solubility greatly limit its application. Accordingly, this study prepared eugenol nanoemulsions by a high-speed shearing technique. Through visual inspection and a series of characterizations, including dynamic light scattering, and confocal laser scanning microscopy, the optimized formula was determined to be 5% (w/w) oil phase (eugenol) and 8% (w/w) surfactant (Tween-80), and the optimized shearing time was 5 min. The optimized nanoemulsion had good stability, small droplets (85 nm), and uniform distribution. At a concentration of 0.02 mg μL⁻¹, the nanoemulsion showed strong inhibition against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Scanning electron microscopy (SEM) images showed severe deformation and membrane rupture of both bacteria treated by the nanoemulsion. This result was further confirmed by the leakage of proteins in both bacteria after treatment. The results of reactive oxygen species (ROS) and malondialdehyde (MDA) measurements indicated that the increased levels of ROS in both bacteria treated by the nanoemulsion triggered lipid peroxidation, thus increasing the MDA levels, ultimately causing changes in cell membrane permeability and disruption of the membrane structure. In addition, the nanoemulsion had a small effect on the proliferation and apoptosis of hepatocytes (L02) and lung cells (BEAS-2B), indicating its good biocompatibility. In this study, we developed a novel eugenol nanoemulsion with high stability and good biological activity, which may provide a promising and effective method for wound treatment in the healthcare area.