Objective To investigate the in vitro inhibitory effect of quaternized chitosan against Klebsiella pneumoniae. Methods Plating serial dilutions were used to detect the direct inhibitory effects of chitosan and quaternized chitosan against Klebsiella pneumoniae. Real-time PCR was employed to detect the expression of important biofilm formation related genes lsrR, lsrK and gmd. Crystal violet staining was utilized to examine the biofilm formation of Klebsiella pneumoniae. Results There were significant decrease in the live bacteria counts in chitosan and quaternized chitosan wells from 6 h after treatment (P＜0.05), and the live bacteria counts in quaternized chitosan wells were significantly lower than those in chitosan wells at 12 h after treatment, with the 12h inhibitory ratios of chitosan and quaternized chitosan of (50.2±9.3)% and (72.6±8.0)% respectively （P＜0.05）. Chitosan and quaternized chitosan increased the expression of lsrR gene, quaternized chitosan decreased the expression of lsrK gene, and there was also a downward trend of expression of gmd gene after treatment with quaternized chitosan. Chitosan and quaternized chitosan achieved an inhibition on the biofilm formation of Klebsiella pneumoniae from 6 h after treatment (P＜0.05), while there was no significant difference in the biofilm formation inhibition ability between chitosan and quaternized chitosan （P>0.05）. Conclusion Chitosan and quaternized chitosan can effectively inhibit the proliferation and biofilm formation of Klebsiella pneumoniae. The antibacterial activity against Klebsiella pneumoniae from quaternized chitosan is better than that of chitosan, and the expression of multiple biofilm formation related genes is influenced by quaternized chitosan.