Composite films of chitosan, fish gelatin and microbial transglutaminase (MTgase) were developed. Films were produced by the casting method and dried at room temperature for 30 h, conditioned for 7 days at 30 °C at a relative humidity (RH) from 11 to 90%, and characterized. Chitosan:fish gelatin films in different proportions (100:0, 75:25, 50:50) with MTgase, were subjected to tensile properties and water vapor transmission (WVT) testing. The results showed that tensile strength decreased with an increase in RH and with an increase in gelatin content. Percent of elongation also increased with increasing RH and gelatin concentration. Water vapor transmission showed an increase proportional to an increase in RH with the presence of gelatin being unfavorable for reducing WVT. Results in this work allowed studying the effect of relative humidity on tensile and water vapor properties of chitosan and fish gelatin films.