Chitosan is widely recognized as a. biodegradable material with broad potential. In order to synthesize chitosan derivatives with quaternary ammonium salt having different methylene spacers, we introduced alkyl groups were selectively introduced to the amine groups of chitosan: N-alkyl chitosan derivatives were quaternized with methyl iodide. Antibacterial activities of resulting water-soluble cationic polyelectrolytes, novel chitosan derivatives, against Staphylococcus aureus (S. aureus) (ATCC 6538P) and Escherichia coli (E. coli) (ATCC14339), gram-positive and gram-negative bacteria, respectively, were evaluated by the viable cell counting method in acetate buffer (pH 6.0). The antibacterial activities of the chitosan derivatives with quaternary ammonium salt increased with an increasing in the chain length of the alkyl substituent, and this increased activity was attributed to the contribution of the increased hydrophobic properties of the derivatives. These results clearly demonstrated that the hydrophobicity and cationic charge of the introduced substituent and the chain movement of chitosan derivatives strongly affect the antibacterial activity of quaternized chitosan derivatives against S. aureus and E. coli.