A novel chitosan antibacterial dispersion system was prepared by oleoyl-chitosan (OCS) nanoparticles (OCNP). We further investigated the antimicrobial mode of OCNP against Escherichia coli and Staphylococcus aureus using a combination of approaches, including measurement of the effect of lecithin and phosphate groups, the conformation of membrane protein, internalization of fluorescein isothiocyanate (FITC)-labeled OCS nanoparticles (FITC-OCS nanoparticles) observed under fluorescence microscopy and DNA/RNA binding assay. Results of fluorescence experiments indicated that OCNP influenced the structure of bacterial membranes. The lecithin effect showed that OCNP bound to cytoplasmic membrane phospholipids of S. aureus, and phosphate groups played an important role. Fluorescence microscopy observations demonstrated that the way OCNP entered into bacteria varied against strains. The gel-retardation experiment showed that OCNP bound strongly to DNA/RNA and retarded their migration in the gels in a concentration-dependent manner. These results indicate that OCNP exerts its antibacterial activity by damaging the structures of cell membrane and putative binding to extracellular targets such as phosphate groups or intracellular targets such as DNA and RNA.