N-(sodium 2-hydroxypropylsulfonate) chitosan (HSCS), N-sulfonate chitosan (SCS) and N-isonicotinic sulfonate chitosan (ISCS) were prepared. The structures of the prepared chitosan derivatives were characterized by nuclear magnetic resonance (H NMR) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, ultraviolet-visible (UV-vis) spectroscopy and elemental analysis (EA). Antibacterial and antibiofilm activities of these chitosan derivatives were evaluated in vitro. The minimum inhibitory concentration (MIC) of HSCS and SCS against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were 0.625 mg/mL and 0.156 mg/mL, respectively. ISCS exhibited MIC values of 0.313 mg/mL and 0.078 mg/mL against E. coli and S. aureus, respectively. ISCS demonstrated superior antibacterial and antibiofilm properties compared to SCS and HSCS. These findings suggest that the incorporation of a pyridine structure into sulfonate chitosan enhances its antibacterial and antibiofilm activities, and the prepared ISCS has a promising application prospect for controlling the reproduction of microorganisms in the field of food packaging.