The interest in developing new materials intended for food packaging based on bacterial cellulose is growing in the recent years. Flexible and transparent films from bacterial cellulose-chitosan-polyvinyl alcohol have shown excellent UV-barrier properties. However, this material interacts with ambient moisture modifying its water activity due to its hydrophilic nature. In this work, a study was carried out to evaluate the changes caused by the water activity. Results showed a plasticizing effect of water molecules increasing the water vapor permeability of the samples from 1.86 × 10-12 to 1.17 × 10-11 g/m·s·Pa, percentage of elongation from 3.25 to 36.55%, and distance to burst from 0.64 to 5.12 mm. The increase of the water activity decreased the Young's modulus and tensile strength. The values of the UV-barrier were maintained at the wide range of water activity. Consequently, water molecules do not affect the UV-barrier properties of the films.