Side effects of the drugs’ oral administration led us to examine the possibility of using diclofenac sodium (DLF) in a polymeric drug delivery system based on electrospun polyacrylonitrile (PAN) nanofibers, which can be produced cost-effectively and with good applicability for transdermal treatments. The inclusion of DLF in PAN nanofibers increased the nanofiber diameter from ~200 nm to ~500 nm. This increase can be attributed to the increase in the spinning solution viscosity. FTIR spectra confirm the entrapment of the DLF into the PAN nanofibers. X-ray diffraction pattern showed that the inclusion of the DLF in the PAN nanofibers had caused the misalignment in the polymeric chains of the PAN, thus resulting in a decrease of the peak intensity at 2θ = 17o. The DLF loaded PAN nanofibers were efficient against the gram-positive Staphylococcus aureus (S. aureus) and gram-negative Escherichia coli (E. coli), with maximum inhibition zone of 16 ± 0.46 mm for E. coli and 15.5 ± 0.28 mm for S. aureus. Good cell viability ~95% for L929 cells in more extended incubation periods was reported. A gradual release of DLF from the PAN nanofiber was observed and can be attributed to the stability of Pan in PBS medium. Cell adhesion micrographs show that cell-cell interaction is stronger than the cell-material interaction. This type of weak cell interaction with the wound dressing is particularly advantageous, as this will not disturb the wound surface during the nursing of the wound.