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Clove oil has a high eugenol content, making it an effective antimicrobial essential oil; nevertheless, its low water solubility, high volatility, and organoleptic qualities limit its use in food systems. As a result, we created an antibacterial system using clove oil-in-water nanoemulsion. Clove oil nanoemulsions were produced using whey protein concentrate (0.1–1%) as an emulsifier by ultrasonication and various physico-chemical characteristics (stability, particle size, zeta-potential, and poly dispersity index) were investigated. Mean particle size, zeta potential and polydispersity index of the most stable nanoemulsion were 279.0 ± 8.43 nm, -34.5 ± 0.12 mV, and 0.179 ± 0.012, respectively. Most stable nanoemulsion was fairly stable at different processing parameters such as various pH (3.0 – 7.0), temperature ranges (63 – 121 °C), and ionic strengths (0.1 – 1.0 M NaCl). Finally, antimicrobial activities, such as minimum inhibitory concentration was found with 50 µL, whereas minimum bactericidal concentration was observed to be 90 µL after 8 h contact time, against E. coli and B. subtilis strains.