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Electrospun nanofibrous mats based on biopolymers have been widely investigated for tissue engineering in recent years, primarily due to remarkable morphological similarity to the natural extracellular matrix (ECM). In this research, electrospun PVA/Chitosan/Starch nanofibrous mats were fabricated using electrospinning method for wound dressing application. The prepared nanofibrous mats were then cross-linked to enhanced the water resistance and also optimize the biodegradation rate followed by characterization and evaluation of their properties as wound dressings. The morphological studies performed by SEM and AFM showed that uniform bead-free electrospun nanofibrous mats were formed. The structural properties of the fabricated mats were characterized by FTIR. The proper porosity and balanced water absorption and water vapor transmission rate (WVTR) of obtained dressings, demonstrate their ability in providing suitable moist environment for wound, result in the appropriate wound breathing and simultaneously efficient handling of wound exudates. Suitable mechanical properties of nanofibrous dressing in both dry and wet states confirm the capability of fabricated wound dressing to protect wound area against the external forces during the healing process. Antibacterial test revealed excellent antibacterial activity of nanofibrous mats against both gram negative and gram positive bacteria. Furthermore, the in vitro cytotoxicity evaluated by MTT assay, proved appropriate cytocompatibility and cell viability of the developed nanofibrous mats which were also verified with in vitro wound healing analysis performed by scratch assay, confirming the remarkable potential of the investigated nanofibrous mats for wound dressing application.