A series of 2-hydroxypropyltrimethyl ammonium chloride chitosan (HACC) was prepared by the reaction of chitosan with glycidyl trimethyl ammonium chloride. Structure of HACC was characterized by FT IR and 1H NMR spectroscopies, and it was proved that substitution reaction mainly occurs on the N element. Antimicrobial activities of HACC was examined against S. aureus, E. coli, and A. niger. Results indicatd that the inhibitory effects of HACC solutions were varied with HACC concentration, quaternization degrees, pH values, metal ions, and heat treatment. The antimicrobial properties of handsheets prepared from HACC were studied by the inhibition zone method, and the sheets had good antimicrobial properties against S. aureus and E. coli, and low inhibition rate against A. niger.