In this research, the influence of the processing conditions, the electrospun coating thickness and ageing on the water vapour and oxygen barrier properties and on the mechanical, morphological and optical properties of the materials have been evaluated. The effect of relative humidity on the mechanical properties was also evaluated. When coating the thermoplastic Wheat gluten film with annealed polyhydroxybutyrate electrospun fibres, water permeability dropped up to ca. 88%. Oxygen permeability was also improved and both water vapour and oxygen barrier properties of the multilayer systems remained over the storage time. The applied outer layers seemed to improve the mechanical performance (increasing the Elastic Modulus and Tensile Strength values) of the plasticized WG film. An increase in water content caused a decrease in Tensile Strength and Elastic Modulus values but an increase in EAB of the multilayer WG film due to the plasticization effect of water. The stretchability of these multilayer systems was greatly affected by the thickness of the outer layer and by the relative humidity although no significant variations were observed due to storage time. The appearance of the thermo-formed wheat gluten films was brown and translucent and it was slightly diminished over the storage time. This work has demonstrated that it is possible to produce water resistant thermoplastic wheat gluten films by combining an inner layer based on wheat gluten with electrospun PHB outer layers. The use of electrospinning was crucial to guarantee a good adhesion between layers which was, in fact, a key factor for the improved barrier properties attained. (C) 2014 Elsevier Ltd. All rights reserved.