The effect of temperature (10–30 °C), relative humidity (RH, 50–90%), and storage period (2–10 days) were studied on the production and sustainability of thiocyanate (SCN − ) and hypothiocyanite–hypoiodite (OSCN − , OI − ) components of a lactoperoxidase system (LPOSI) in chitosan film by applying response surface methodology and Doelhert experimental design. The results of analysis of variance indicated that the temperature and storage period exhibited a significant effect on the film properties. Temperature had a positive influence on OSCN − , OI − , while these products (OSCN − , OI − ) decreased during the storage period, and SCN − disappeared after 2 days. Lactoperoxidase systems with iodine (LPOSI) or without iodine (LPOS), incorporated into chitosan films, showed inhibition of Xanthomonas campestris pv. Mangiferae indicae and did not changed the chitosan film permeability to gas and water vapor.