A novel amphipathic chitosan derivative, O-acetyl-chitosan acetic ester (ACHA), was synthesized by the reaction of chitosan with acetic acid in the presence of thionyl chloride. The physicochemical properties of ACHA were characterized by FTIR, 1 H NMR, TGA, and XRD. The yield (Y) of ACHA was 79.4%, and the degree of acetylation (DA) of ACHA was 1.04. Compared to CS, ACHA could be dissolved in many organic solvents, deionized water, and aqueous solution. Our results showed that ACHA exhibited a superior antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. These findings indicated that ACHA was preferable for use as antimicrobial agents in wound healing, food preservative, and tissue engineering.