The objectives of the current study were to prepare and characterise cinnamon essential oil nanoemulsion (NCEO) and investigate its antibacterial activity in vitro and milk. The average size of NCEO droplets was 66.6 nm. NCEO was overall more effective than free essential oil on Listeria monocytogenes in milk stored at 7 °C/6 days, whereas Escherichia coli was more sensitive to essential oil than NCEO in milk samples stored at 7 and 37 °C. The type of bacterial strain, NCEO concentration and storage temperature appear to have various impacts on the antibacterial activity of NCEO in milk.