The antimicrobial activities of lemon oil based nanoemulsion and two different concentrations of lemon essential oil (100% and 10%) on food-brne pathogens (Staphylococcus aureus, Klebsiella pneumoniae, Enterococcus faecalis and Salmonella Paratyphi A) and fish spoilage bacteria (Photobacterium damselae, Enterococcus faecalis, Vibrio vulnificus, Proteus mirabilis, Serratia liquefaciens, and Pseudomonas luteola) were compared in terms of disc diffusion, minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC). The constitutes of extracted lemon essential oil were identified by using GC-MS. Viscosity, the mean droplet size, thermodynamic stability and refractive index of nanoemulsions were determined. The main components detected in the lemon essential oil were d-limonene, p-cymene, β-pinene with percentages of 52.85%, 14.36%, and 13.69%, respectively. It was found that lemon nanoemulsion was more effective on food-borne pathogens except K. pneumoniae than 100% lemon essential oil. 10% lemon essential oil showed the highest inhibition effect on S. Paratyphi A. The conversion of the essential oil into nanoemulsion improved antimicrobial activity. According to value of MIC, both nanoemulsion and 100% essential oil inhibited bacterial growth of all of the pathogen bacteria tested whereas they were less effective on inhibition of fish spoilage bacteria. However, 10% essential oil was more effective on spoilage bacteria than pathogens. MBC showed that nanoemulsion and 100% lemon essential oil presented a noticeable bactericidal activity against S. paratyphi A whereas 10% lemon essential oil was found as ≥25 mg/mL against pathogens and spoilage bacteria. Therefore, the use of nanoemulsion based on lemon essential oil can have potential as a natural antimicrobial agent against food-borne pathogen and spoilage bacteria for fish processing industry.