Abstract: To address the application limitations of eugenol due to its poor water solubility and volatility, this study developed a novel delivery system based on an ultrasound-assisted whey protein isolate-low methoxyl pectin bilayer nanoemulsion (WPI-LMP BN). Box-Behnken design was employed to optimize key parameters (WPI/LMP concentrations, ultrasound power/time), with mean particle size and polydispersity index (PDI) as critical evaluation indicators. The microscopic morphology and intermolecular interactions of the emulsion were analyzed using scanning electron microscopy and Fourier transform infrared spectroscopy; its stability under pH, ionic strength, and temperature stress was systematically evaluated; and finally, agar diffusion assays were employed to compare its antibacterial activity with that of free eugenol. The optimal conditions were established as follows: 2.60% WPI, 1.20% LMP, 500 W ultrasound power, and 8 min ultrasound time. Under these conditions, the resulting WPI-LMP BN exhibited a particle size of 318.6 nm, a PDI of 0.227, and an encapsulation efficiency of 76.53%. Scanning electron microscopy revealed spherical and uniformly distributed droplets, while Fourier transform infrared spectroscopy confirmed the successful formation of a WPI-LMP composite interface. Stability studies indicated that the nanoemulsion maintained good physical stability under various environmental stresses, including a broad pH range (3~10), Na⁺ concentrations up to 250 mmol/L, and temperatures up to 80 ℃. Antibacterial assays showed that the inhibition zone diameters of WPI-LMP BN against Staphylococcus aureus and Escherichia coli were significantly increased by 43.11% and 41.81%, respectively, compared to free eugenol, demonstrating a substantial enhancement in antimicrobial efficacy. These findings indicate that the ultrasound-assisted WPI-LMP BN possesses excellent stability, effectively encapsulates eugenol, and markedly improves its antibacterial performance, offering a promising approach for developing stable and efficient plant essential oil-based antimicrobial delivery systems.