Abstract Diabetic wounds are susceptible to microbial infection. The treatment of these wounds requires a higher payload of growth factors. With this in mind, the strategy for this study was to utilize a novel payload comprising of Eudragit RL/RS 100 nanofibers carrying the bacterial inhibitor gentamicin sulfate (GS) in concert with recombinant human epidermal growth factor (rhEGF); an accelerator of wound healing. GS containing Eudragit was electrospun to yield nanofiber scaffolds, which were further modified by covalent immobilization of rhEGF to their surface. This novel fabricated nanoscaffold was characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, and X‐ray diffraction. The thermal behavior of the nanoscaffold was determined using thermogravimetric analysis and differential scanning calorimetry. In the in vitro antibacterial assays, the nanoscaffolds exhibited comparable antibacterial activity to pure gentemicin powder. In vivo work using female C57/BL6 mice, the nanoscaffolds induced faster wound healing activity in dorsal wounds compared to the control. The paradigm in this study presents a robust in vivo model to enhance the applicability of drug delivery systems in wound healing applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 641–651, 2018.