© 2020 Elsevier LtdIn recent years, electrospun nanofiber mats based on biopolymers have been extensively investigated for tissue and biomedical engineering, mainly because of remarkable morphological similarity with the natural extracellular matrix. The current study focuses on the preparation of novel natural spider silk (SS) embedded Poly(vinyl alcohol)(PVA)/Sodium alginate (NaAlg) wound dressings with desirable properties for a wound dressing application. The nanofibers' surface morphology and structure were observed by a field emission scanning electron microscope (FE-SEM). In-vivo evaluation of PVA/NaAlg based electrospun nanofiber mats as a wound dressing material and their comparison to commercially available wound dressings was carried out by in-vivo tests on rabbit models. Some morphometric parameters such as counting cells, blood vessels, endothelial cells, determination of the area of the wound closure, wound healing performance, speed of wound healing and collagen thickness were investigated using OM and FE-SEM post-processing. Furthermore, in-vitro biocompatibility and cellular behavior such as adhesion and proliferation of mouse fibroblast cells (L929) were studied by XTT assay on developed nanofiber mats. The results of this experimental study indicated that the natural spider silk embedded electrospun nanofiber mat (PVA/NaAlg/SS) accelerated the rate of wound healing compared to other groups by improving the collagen formation rate, proliferative cell activity as well as decreasing the inflammatory cell amount. Furthermore, the results of the in-vivo and in-vitro experiments suggest that novel PVA/NaAlg/SS nanofiber mats might be a fascinating bioactive wound dressing for clinical applications.