The nano-emulsion of cinnamon bark oil (CBON) was formulated and investigated for stability and antimicrobial properties. The requisite hydrophilic lipophilic balance (rHLB) and surfactant concentration (Smix) of CBON were found to be 10 and 15%, respectively. A nano-emulsion prepared using 10 g/100 mL CBO and 15 g/100 mL Smix at ultrasonication for 2 min resulted in the smallest droplet size and polydispersity index (PDI) (d = 94.51 ± 0.19 PDI = 0.283 ± 0.009). The droplet size and PDI of the CBO was found to be influenced by HLB value, treatment time, and smix concentration. This study also aimed to evaluate the antimicrobial properties of the prepared oil-in water nano-emulsion of cinnamon oil against E. coli, S. aureus, and S. cerevisiae. The CBON were found to be more efficient in inhibiting and inactivating microorganisms than the crude CBO. The sensitivity of microorganism towards CBON depend on the type of microorganism with S. cerevisiae being the most sensitive followed by E. coli and S. aureus. S. aureus were inhibited with a complete 6 log reduction within 60 min of incubation with CBON. Whereas in the case of E. coli, and S. aureus a reduction of 6 log CFU and 5 log CFU respectively were reported after 120 min of exposure.