Shubham Kanaujiya,1,* Dilip Kumar Arya,1,* Prashant Pandey,1 Sneha Singh,2 Giriraj Pandey,1 Shabnam Anjum,3 Md Meraj Anjum,1 Daoud Ali,4 Saud Alarifi,4 Vijayakumar MR,1 Sri Sivakumar,2 Saurabh Srivastava,5 PS Rajinikanth1 1Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow, India; 2Department of Chemical Engineering, IIT Kanpur, Kanpur, India; 3Department of Tissue Engineering, School of Intelligent Medicine, China Medical University, Shenyang, Liaoning, 110122, People’s Republic of China; 4Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia; 5Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Hyderabad, Telangana, India*These authors contributed equally to this workCorrespondence: PS Rajinikanth, Department of Pharmaceutical Sciences, Babasaheb Bhimrao Ambedkar University, Lucknow, India, Email psrajinikanth222@gmail.comBackground: The healing of burn wounds is a complicated physiological process that involves several stages, including haemostasis, inflammation, proliferation, and remodelling to rebuild the skin and subcutaneous tissue integrity. Recent advancements in nanomaterials, especially nanofibers, have opened a new way for efficient healing of wounds due to burning or other injuries.Methods: This study aims to develop and characterize collagen-decorated, bilayered electrospun nanofibrous mats composed of PVP and PVA loaded with Resveratrol (RSV) and Ampicillin (AMP) to accelerate burn wound healing and tissue repair.Results: Nanofibers with smooth surfaces and web-like structures with diameters ranging from 200 to 400 nm were successfully produced by electrospinning. These fibres exhibited excellent in vitro properties, including the ability to absorb wound exudates and undergo biodegradation over a two-week period. Additionally, these nanofibers demonstrated sustained and controlled release of encapsulated Resveratrol (RSV) and Ampicillin (AMP) through in vitro release studies. The zone of inhibition (ZOI) of PVP-PVA-RSV-AMP nanofibers against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was found 31± 0.09 mm and 12± 0.03, respectively, which was significantly higher as compared to positive control. Similarly, the biofilm study confirmed the significant reduction in the formation of biofilms in nanofiber-treated group against both S. aureus and E. coli. X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) analysis proved the encapsulation of RSV and AMP successfully into nanofibers and their compatibility. Haemolysis assay (%) showed no significant haemolysis (less than 5%) in nanofiber-treated groups, confirmed their cytocompatibility with red blood cells (RBCs). Cell viability assay and cell adhesion on HaCaT cells showed increased cell proliferation, indicating its biocompatibility as well as non-toxic properties. Results of the in-vivo experiments on a burn wound model demonstrated potential burn wound healing in rats confirmed by H&E-stained images and also improved the collagen synthesis in nanofibers-treated groups evidenced by Masson-trichrome staining. The ELISA assay clearly indicated the efficient downregulation of TNF-alpha and IL-6 inflammatory biomarkers after treatment with nanofibers on day 10.Conclusion: The RSV and AMP-loaded nanofiber mats, developed in this study, expedite burn wound healing through their multifaceted approach. Keywords: electrospinning, nanofiber, resveratrol, ampicillin, burn wound healing, collagen