Methylated N-(3-pyridylmethyl) chitosan chloride (M3-PyMeChC) with various degrees ofN-substitution (DS), degrees ofquaternization (DQ), and molecular weights were synthesized by single methylation ofN-(3-pyridylmethyl) chitosan with iodomethane under the basic condition. The DS and DQ were calculated by 1H NMR, and they were in a range of 35 ± 1%-80 ± 2% and 72 ± 1%-80 ± 2%, respectively. Depolymerization of the methylated chitosan derivatives was carried out by using 0.1-0.5% (w/v) of sodium nitrite solution and their molecular weights were determined by gel permeation chromatography. It was found that the concentration of sodium nitrite solution increased with a decrease in molecular weight. Bactericidal activity of these M3-PyMeChCs were determined by using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods against Escherichia coli ATCC 25922 (Gram-negative) and Staphylococcus aureus ATCC 6538 (Gram-positive) bacteria compared to the N,N,N-trimethyl chitosan chloride (TMChC) in accordance with the standard method of the National Committee for Clinical Laboratory Standards (NCCLS). It was found that the bactericidal activity was dependent on the chemical structure of quaternary ammonium moiety and the molecular weight, at constant DQ. The result revealed that the N,N,N-trimethyl ammonium group showed higher bactericidal activity than the N-methylpyridinium group at similar DQ and molecular weight. Moreover, the bactericidal activity of M3-PyMeChC tended to decrease with decreasing molecular weight from 82 kDa to 8 kDa, at DQ of 80 ± 2%.