The positionally substituted 2′-O-hydroxypropyl trimethyl ammonium chloride chitin (2′-O-HTACCT) with different substitution degree of quaternary ammonium salt were synthesized directly from β-chitin, after that these 2′-O-HTACCT were then deacetylated to obtain corresponding 2′-O-hydroxypropyl trimethyl ammonium chloride chitosan (2′-O-HTACCS) with different deacetylation degree. The antibacterial activity and mechanism against E. coli and S. aureus of these 2′-O-HTACCT and 2′-O-HTACCS were investigated with various techniques. The results demonstrated that the 2′-O-HTACCT had antibacterial effect against S. aureus, but it had negligible antibacterial effect against E. coli, maybe due to the lack of anionic compound such as teichoic acid on the E. coli’s cell wall. Impressively, the high antibacterial effect against E. coli of the 2′-O-HTACCS was derived only from its -NH₂, but the ultra high antibacterial effect against S. aureus of the 2′-O-HTACCS was derived both from its -NH₂ and -N⁺(CH₃)₃, and enhanced as its pH value decreased. Thus, the positionally substituted 2′-O-HTACCS played an important role as a high-performance antibacterial bio-resource polymer against E. coli and S. aureus.