Using ultrasonic technology, trans-cinnamaldehyde as a natural antibacterial compound was used to prepare nano size emulsions to increase its bioavailability and therefore bactericidal action. Nanoemulsions containing trans-cinnamaldehyde as an active agent and 1,8 cineol as co additive oil (Ostwald ripening inhibitor) were formulated using probe sonicator. Three different determining factors, namely time of sonication, surfactant to oil ratio and type of emulsifier (Tween 80 and Tween 20) were investigated to enhance the stability profile. In addition, the effect of changes in the particle size and emulsifier on the antibacterial activity against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were examined using agar dilution method. Then, the effect of optimized formulation on the membrane fluidity and cell constituent release, were investigated by analysis of membrane lipids using GC-MS and IR spectrometry, respectively. The data showed that a 15min sonication of the formulation containing Tween 80 as emulsifier with surfactant to oil ratio of 2:1 (w/w) resulted in a significant stability for 6months with considerably small particle size of 27.76±0.37nm. Furthermore, the nanoemulsion showed great antibacterial activity and could reduce the minimum inhibitory concentration (MIC) from 8 to 1mg/mL against E. coli and S. aureus, and from 16 to 2mg/mL against P. aeruginosa. Interestingly, E. coli's membrane fluidity increased dramatically after treatment with the optimum nanoemulsion (T804). This study revealed that nanoemulsion of trans-cinnamaldehyde and 1,8 cineol has substantial antibacterial activity against selected microorganisms.