Recently, the number of patients infected by drug-resistant pathogenic microbes has increased remarkably worldwide, and a number of studies have reported new antibiotics from natural sources. Among them, chitosan, with a high molecular weight and -conformation, exhibits potent antimicrobial activity, but useful applications as an antibiotic are limited by its cytotoxicity and insolubility at physiological pH. In the present study, the antibacterial activity of low molecular weight water-soluble (LMWS) -chitosan (1k, 5k, and 10k with molecular masses of 1, 5, and 10 kDa, respectively) and -chitosan (1k, 5k, and 10k) was compared using a range of pathogenic bacteria containing drug-resistant bacteria isolated from patients at different pH. Interestingly, 5k and 10k exhibited potent antibacterial activity, even at pH 7.4, whereas only 10k was effective at pH 7.4. The active target of -chitosan is the bacterial membrane, where the leakage of calcein is induced in artificial PE/PG vesicles, bacterial mimetic membrane. Moreover, scanning electron microscopy showed that they caused significant morphological changes on the bacterial surfaces. An in vivo study utilizing a bacteria-infected mouse model found that LMWS -chitosan could be used as a candidate in anti-infective or wound healing therapeutic applications.