Clove oil is an essential oil used as a biodegradable antibacterial agent in food and pharmaceutical products. Emulsions are used to stabilize and increase the antimicrobial efficacy of oils in aqueous solutions. Smaller droplets provide better distribution of the oil. In this study, we formulate clove oil in aqueous solution by nanoemulsion. The aim of this study was to optimize key parameters in the preparation of clove oil nanoemulsion to obtain stable emulsions with less than 50 nm droplet size. The evaluated parameters include the concentration of clove oil, concentration of emulsifier, hydrophilic–lipophilic balance (HLB) number, and ultrasonication time. We determined the following response parameters: droplet size, polydispersity index, and zeta potential, and we aimed to achieve <50 nm droplet size with low polydispersity index and a highly negative zeta potential. The optimization and evaluation was obtained via Taguchi method. Taguchi method identified the concentration of clove oil and the HLB as the most affecting factors in the preparation of nanoemulsions. The optimum conditions for the preparation of nanoemuslion such as mean droplet size, polydispersity index, and zeta potential were 50 nm, 0.49 and −40.7 mV, respectively. This formulation of clove oil resulted in minimum inhibitory concentration against Escherichia coli and Bacillus cereus of 16 and 32 μg/ml, respectively, and minimal bactericidal concentrations of 16 and 64 μg/ml, respectively.