Cinnamon oil (extracted from Cinnamomum zeylanicum) nanoemulsion was formulated using Tween 80 and water by ultrasonic emulsification. Process of nanoemulsion formulation was optimized for parameters such as surfactant concentration, oil-surfactant mixing ratio and emulsification time. Surfactant concentration was found to be inversely related to droplet size and directly related to stability. Increase in emulsification time resulted in decrease in droplet diameter. Stable cinnamon oil formulation (CF3) having droplet diameter of 65 nm was formulated after sonication for 30 min. Formulated nanoemulsion was evaluated for bactericidal efficacy against Bacillus cereus. Time and concentration dependent killing of B. cereus cells was observed upon treatment with nanoemulsion. Even at a higher dilution of CF3, significant reduction in bacterial population was observed. Alteration in membrane permeability of interacted samples was suggested by quantifying the release of UV absorbing materials. Bacterial staining with acridine orange/ethidium bromide supported kinetics of killing data and also substantiated the above findings of alteration in membrane permeability. FTIR illustrated disappearance of peak corresponding phosphate vibration at 1078 cm-1 and 536 cm-1, and peak associated with vibration of acyl chains of lipid at 2852 cm-1 was shifted to 2854 cm-1 which suggested deformation of membrane phospholipids in nanoemulsion treated cells. SEM observations demonstrated membrane distortion leading to cell lysis. These results propose the potential use of cinnamon oil nanoemulsion for preservation of minimally processed food. Copyright © 2013 American Scientific Publishers All rights reserved.