The purpose of this research was to develop a chitosan sulfobetaine (CS-SNCC) film via the solution-casting method as a biodegradable antibacterial material for biomedical applications. Chitosan and monochloro-triazine sulfobetaine were used as the raw materials for CS-SNCC preparation, and Fourier-transform infrared (FTIR), ultraviolet-visible (UV-Vis), energy-dispersive X-ray (EDX), and X-ray photoelectron spectroscopy (XPS) spectra were used to characterize and analyze the structure of the synthesized CS-SNCC. Furthermore, the swelling property, thermal stability, biodegradability, cytocompatibility, and antibacterial properties of the CS-SNCC film were comprehensively investigated and compared with those of the chitosan film. The results for the film’s enzymatic biodegradation behavior show that the CS-SNCC film undergoes a weight loss of 45.54% after 21 days of incubation. In addition, the CS-SNCC film effectively resists bacterial adhesion, prevents the formation of bacteria biofilms, and exhibits high antibacterial activity, with inactivation rates of 93.43% for Escherichia coli and 91.00% for Staphylococcus aureus. Moreover, the CS-SNCC film shows good cellular activity and cytocompatibility according to the cytotoxicity results. Therefore, the prepared biodegradable, cytocompatible, antibacterial, and biofilm-controlling CS-SNCC film has potential for biomedical applications.