This work aims to prepare and characterize one kind of nitric oxide (NO)-releasing conjugation of quaternary ammonium salt to chitosan, as well as to evaluate the anti-bacterial properties of diazeniumdiolates and the changes in NO release properties. The newly synthesized diazeniumdiolates are obtained from glycidyl-trimethyl-ammonium chloride (GTMAC)-bearing chitosan derivatives (HTCC) with different molecular weights (280 and 670 KDa) and are used as NO donor species. An HTCC with high molecular weight (670 KDa) exhibits higher storage capacity for NO (up to 357.70 nmol NO/mg) than one with a low molecular weight (280 kDa). The NO release durations (7 h) observed for the HTCC diazeniumliolates with higher molecular weight (670 kDa) was slightly higher than that of HTCC diazeniumliolates with lower molecular weight (280 kDa). By determining the inhibition zone diameter, HTCC-NO with lower molecular weight (280 kDa) showed significantly higher inhibition capabilities against E. coli than HTCC, crude chitosan, and water control.