Recently, layered structures composed of nanofibers have gained attention as a novel material to mimic skin tissue in wound healing applications. The aim of this study is to develop a novel hybrid bilayer material composed of zein based composite film and nanofiber layers as a wound dressing material. The upper layer was composed of H. perforatum oil incorporated zein film including MMT and the bottom layer was comprised of 3D electrospun zein/MMT nanofibers to induce wound healing with the controlled release of H. perforatum oil. The bilayer composites were characterized in terms of mechanical test, WVP, water uptake and surface wettability. Antimicrobial activity of the wound dressings against microorganisms were investigated by disc diffusion method. In vitro cytotoxicity of monolayer film and bilayer structure was performed using WST-1 assay on HS2 keratinocyte and 3T3 cell lines. Results indicated that the prepared monolayer films showed appropriate mechanical and gas barrier properties and surface wettability for wound healing. Controlled release of H. perforatum oil was obtained from fabricated membranes up to 48 h. Bilayer membranes showed antimicrobial activity against E. coli, S. aureus, and C. albicans and did not show any toxic effect on NIH3T3 mouse fibroblast and HS2 keratinocyte cell lines. In vitro scratch assay results indicated that H. perforatum oil had a wound healing effect by inducing fibroblast migration. The proliferation study supported these results by increasing fibroblast proliferation on H. perforatum oil loaded bilayer membranes.