Because no known pure biopolymer exhibits all the desired physicochemical properties required for every conceivable packaging application, multilayer films are here proposed as an alternative to improve the performance of protein-based films. In this work, three-layer films based on plasticized wheat gluten films have been developed by applying a more hydrophobic electrospun polyhydroxyalkanoate layers on both sides of the protein film. A commercial polyhydroxybutyrate (PHB) and a polyhydroxybutyrate-co-valerate copolymer with 3 % valerate content (PHBV3) have been used. The morphology, oxygen and water vapour barriers, the optical properties as well as the mechanical performance have been characterized. The effect of film processing temperature (145 and 160 °C) and the amount of electrospun material constituting the outer layers played an important role on the final physicochemical properties of these films. The results showed that water vapour barrier properties were mainly governed, on the one hand, by the deposited amount of the electrospun PHB layer and, on the other hand, by the temperature used for PHBV3-WG-PHBV3 assembly. However, oxygen permeability was greatly influenced by the type and amount of the polyhydroxyalkanoates (PHAs) instead of the temperature used. The lowest water vapour permeability (WVP) and oxygen permeability (OP) values were obtained for three-layer films prepared with 1 mg PHBV3·cm−2 and processed at 160 °C, reaching values of 3.14 ± 0.2 e−11 kg·m·Pa−1·s−1·m−2 and 4.36 ± 0.05 e−15 m3·m·m−2·s−1·Pa−1, respectively. The addition of PHB or PHBV3 outer layers improved the mechanical properties (especially Young’s modulus, which increased up to ~40 % for the three-layer films containing thicker layers) of plasticized wheat gluten films. However, the transparency of the three-layer films was slightly affected by the processing temperature and by the thickness of the outer layer.