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Totarol is a natural antimicrobial compound extracted from the heartwood of Podocarpus totara, a conifer native to New Zealand. The effects of whey protein-totarol nanoparticles treated with ultrasound on the physiochemical properties and the growth of Staphylococcus aureus were investigated. The particle size of whey protein-totarol nanoparticles was reduced by ultrasound treatment from 31.24 ± 5.31 to 24.20 ± 4.02 nm, and the size distribution was also narrowed by the treatment. Viscosity and modulus data indicated that the flow behaviors of whey protein-totarol nanoparticles seemed to be Newtonian and exerted a typical viscoelastic fluid at protein content of 15% (w/v). Rheological properties were more insensitive to ultrasonic time. Time-killing assays, agar diffusion tests, the cell membrane damage analysis, and microstructure were exploited to study the antibacterial properties of whey protein-totarol nanoparticles. The MIC of whey protein-totarol nanoparticles after ultrasound treatment decreased from 4 to 2 μg/mL compared with that without ultrasound treatment. Whey protein-totarol nanoparticles treated with ultrasound resulted in a significant (P < 0.05) decrease in time killing after 24 h. The agar diffusion results showed that the inhibition zones of whey protein-totarol nanoparticles were 12 and 36 mm for untreated and treated with ultrasound, respectively. The cell membrane damages and the microstructure changes also proved that whey protein-totarol nanoparticles treated with ultrasound had strong antibacterial activities against S. aureus and that the antibacterial effectiveness enhanced with the increasing of ultrasonic time. These findings suggested that whey protein-totarol nanoparticles treated with ultrasound were more effective against S. aureus than untreated nanoparticles.