Water molecules modify the properties of biodegradable films obtained from hydrophilic materials. Most studies dealing with thermal, mechanical and barrier properties of hydrophilic films are carried out under one relative humidity (RH) condition. The objective of this work was to evaluate the effect of the moisture content on the thermal, mechanical and barrier properties of chitosan films under several RH conditions. Microclimates, obtained with saturated salt solutions were used for conditioning samples and the properties of the films were evaluated under each RH condition. Chitosan films absorbed up to 40% of moisture at the higher RH studied. The percentage of elongation and the water vapour permeability increased while tensile strength, Young's modulus and glass transition temperature decreased, when the moisture content increased. The results suggest that the water molecules plasticized the polymer matrix, changing the properties when the films were in contact with high RH environments.