Verbascum thapsus (V. thpsus), family Scrophulariaceae, has considerable importance in traditional medicine worldwide because of its antioxidant and anti-inflammatory activities. V. thpsus was used in traditional medicines as a useful drug for lung disease, sore throat, wound healing, and treatment of whooping cough. The aim of this study was to extract of V. thpsus bioactive fraction using antibacterial assay guided fractionation methodology and develop a system based on electrospun nanofibrous membrane (NFM) that can be effective by releasing the extract of V. thapsus for antibacterial and wound healing applications. For this purpose, the fractionation of total extract was done using Liquid-Liquid extraction method. The selected fraction based on its anti-bacterial activity was then subjected to the silica gel column chromatography for further purification. Since electrospinning is an economical and relatively simple method to produce continuous and uniform nanofibers, and due to its high specific surface area, adjustable pore size, and flexibility, special attention has been paid to loaded the most effective fraction on PVA nanofibers for applications such as wound dressings. The obtained result showed that, the purified V. Thapsus extract has a concentration-dependent antimicrobial activity against Escherichia coli and Staphylococcus aureus. The phytochemically analyses of bioactive fraction by High-performance liquid chromatography (HPLC) proved the presence of 6 phenolic acids, 4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, and flavonoid, rutin, as the major compounds. Also, physicochemical characterization of PVA-selected extract loaded electrospun nanofibrous membranes (NFM) were analyzed by scanning electron microscope (SEM), Fourier transform infrared spectrometer (FT-IR). MTT and hemolysis assays were done to affirm the biocompatibility of fabricated scaffolds. Release profile of extract loaded- NFM showed continues release of extract from mat during 90h. Moreover, the capability of these NFM in wound healing application was evaluated in-vitro and in-vivo. The cell viability test (MTT), cell adhesion images, antioxidant, antibacterial, hemolysis assays and in-vitro and in-vivo wound healing assays confirmed that fabricated NFM containing 5 % butanolic extract were the most biocompatible scaffold for wound dressing applications and accelerates the rate of wound closure. The obtained outcomes confirmed that V. thpsus/PVA NFM can be considered as promising scaffold for potential wound dressings.