Essential oils of Mentha piperita, Punica granatum, Thymus vulgaris and Citrus limon in olive oil as a carrier were mixed with biopolymer chitosan to prepare nanoemulsions. The formulations were prepared using the following ratios: 0.5:0.5:4, 1:1:4, and 2:3:4 of chitosan: essential oil: olive oil, respectively, representing 12 formulations based on four essential oils. Based on the characterization of nanoemulsions, M. piperita, T. vulgaris, and C. limon oils produced the smallest droplets. However, P. granatum oil produced high droplets size. The products were evaluated in vitro for antimicrobial activity against two pathogenic food bacteria, Escherichia coli and Salmonella typhimunium. The in vivo antibacterial activity was further investigated on minced beef meat during storage at 4 °C for ten days. Based on the MIC values, E. coli was more susceptible than S. typhimunium. Chitosan was more effective as an antibacterial than essential oils (MIC = 500 and 650 mg/L against E. coli and S. typhimunium). Among the tested products, C. limon had a more antibacterial effect. In vivo studies proved that C. limon and its nanoemulsion were the most active products against E.coli. These results suggest that chitosan-essential oil nanoemulsions may help extend the shelf life of meat by acting as antimicrobial agents.