A series of chitosan with different molecular weights obtained by gamma -irradiation depolymerization and another series of deacetylated chitosan were synthesized. Several N,O-carboxymethylated chitosan and O-carboxymethylated chitosan were also produced. The above samples were characterized by Fourier transform infrared spectroscopy (FTIR). Their antibacterial activities against E. coli were explored by the optical density method. The antibacterial activity of chitosan is influenced by its molecular weight, degree of deacetylation, concentration in solution, and pH of the medium. Antibacterial activities were also found to be increased in the order of N,O-carboxymethylated chitosan, chitosan, and O-carboxymethylated chitosan. Fluorescence of the FITC (fluorescein isothiocyanate)-labeled chitosan oligomers at the inside of the E. coli cell was observed by a confocal laser scanning microscope. The antibacterial activity of chitosan oligomers seems to be caused mainly by the inhibition of the transcription from DNA.