The antimicrobial properties of chitosan, a derivative of chitin, were investigated in the solid and liquid culture against bacteria associated with waterborne disease in order to assess the potential for using chitosan as a natural disinfectant. Six strains which included three gram-negative and three gram-positive bacteria were studied. The effects of the deacetylation degree, concentration, and molecular weight of chitosan on antibacterial activities were assessed. Chitosan exhibited the highest antibacterial activity against the Pseudomonas aeruginosa on the solid agar. Similar tendency was found when the bacteria were cultivated in liquid broth. The higher deacetylation degree and higher concentration of chitosan cause the higher antibacterial activities. The effect of molecular weight of chitosan on the inhibition efficacy of bacteria is dependent on the species of bacteria. Escherichia coli is sensitive to chitosan during its death phase and logarithmic phase. The antibacterial mechanism of chitosan was illustrated by the surface charge and persistence length. Results indicated that chitosan is potential as a natural disinfectant.