Six kinds of water-soluble chitosan were prepared by grafting aminofunctionality onto chitosan at the C-6 position, and their antimicrobial activities were investigated against three Gram-negative and three Gram-positive bacteria. Among the derivatives, dimethylaminoethyl-chitosan (DMAEC) has the highest potential to suppress the growth of bacteria. To elucidate detailed antimicrobial modes of action against bacteria, cell integrity, outer membrane (OM), and inner membrane (IM) permeabilization assays were investigated. When treated with DMAEC, the release of 260 nm absorbing materials quickly increased for both Escherichia coli and Staphylococcus aureus, but the absorbance value was different due to the difference in cell structures. In OM and IM permeabilization assays, DMAEC rapidly increased 1-N-phenylnaphthylamine uptake and the release of cytoplasmic beta-galactosidase via an increase in the permeability of OM and IM. Moreover, DMAEC90 prepared from 90% deacetylated chitosan had more activity than DMAEC50 prepared from 50% deacetylated chitosan, and these results revealed that the antimicrobial action of water-soluble chitosans was dependent on the degree of deacetylation and the substituted group.