In this study, cinnamon oil (CIO) was loaded into glycol chitosan-eicosapentaenoic acid nanoparticles (GC-EPA NPs) and their antibacterial effects were evaluated against Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). The GC-EPA conjugate was synthesized through amide bonding and the conjugate could self-assemble into core-shell structure in aqueous solution. The GC-EPA NPs containing CIO (GC-EPA NPs-CIO) were prepared by incorporating CIO into the core of NPs via hydrophobic interaction. The GC-EPA NPs-CIO inhibited cell growth over 95% at 0.3% (w/w) concentration against S. aureus and S. epidermidis. Therefore, it is expected that the GC-EPA NPs-CIO may have a potential for widening the use of CIO in various industrial fields including medicines and cosmetics.