In order to improve mechanical properties and biomedical behavior of chitosan-based polymeric films, the effect of nanocrystalline cellulose (NCC) and calcium peroxide (CP) particles on the properties of polymeric films based on chitosan (CS) and gelatin (GL) were investigated in this study. The films were characterized by Fourier-transform infrared (FTIR), tensile, swelling, water vapor transmission rate (WVTR), antibacterial, oxygen release and cell culture tests. FTIR results indicated that hydrogen bonding has been formed between functional groups of the constituents. The mechanical results showed that the combination of both CP and NCC had better results in improving the mechanical properties of the films. The WVTR and swelling results indicated that CP and NCC particles reduced the amount of WVTR and swelling of the samples. By Adding CP to the film composition, the antibacterial activity of the films against E. coli bacteria increased. The oxygen release for the films containing CP has its maximum value during the first day and it approaches a constant value for 10 days. The MTT assay results revealed that the growth of the human fibroblast cells was increased during 7 days, showing that the chitosan-based films containing CP and NCC had no toxicity and never cause cell death.