To improve the application of essential oils as natural antimicrobial preservatives, the objective of the present study was to determine physical, antimicrobial, and biophysical properties of eugenol after nanoencapsulation by sodium caseinate (NaCas). Emulsions were prepared by mixing eugenol in 20.0 mg/mL NaCas solution at an overall eugenol content of 5.0-137.9 mg/mL using shear homogenization. Stable emulsions were observed up to 38.5 mg/mL eugenol, which had droplet diameters of smaller than 125 nm at pH 5-9 after ambient storage for up to 30 days. The encapsulated eugenol had similar minimal inhibitory and minimal bactericidal concentrations as free eugenol against Escherichia coli O157:H7 ATCC 43895, Listeria monocytogenes Scott A, and Salmonella Enteritidis but showed better inhibition of E. coli O157:H7 than free eugenol during incubation at 37 °C for 48 h. After 20 min interaction at 21 °C, bacteria treated with encapsulated eugenol had a greater reduction of intracellular ATP and a greater increase of extracellular ATP than free eugenol, suggesting the enhanced permeation of eugenol after nanoencapsulation. However, such overall trend was not observed when examining bacterial morphology and uptake of crystal violet, suggesting the possible membrane adaptation. Findings from this study showed the feasibility of preparing nanoemulsions with high loading of eugenol using NaCas.