A novel L-Arginine-copper complex (L-Arg-Cu) was synthesized regarding the necessity for impeding pathogenic microorganisms with no usage of antibiotics. The L-Arg-Cu complex was immobilized onto the graphene oxide surface (GO/L-Arg-Cu). The Fourier transform infrared and Raman spectroscopies, as well as X-ray diffraction, thermogravimetric analysis, field emission scanning electron microscopy, energy-dispersive X-ray (EDX) spectroscopy, were performed to evaluate the chemical interactions and physical properties of the synthesized compound. To achieve better antimicrobial and wound healing properties of electrospun nanofiber (NF) scaffolds, the GO/L-Arg-Cu composite was loaded in polyvinyl alcohol (PVA). It was attempted to improve the stability and mechanical properties of the fabricated NFs via a heat-induced cross-linkage with citric acid (CA). The SEM images analysis revealed the cross-linked PVA/GO/L-Arg-Cu NFs (PVA/GO/L-Arg-Cu/CA NFs) with an average diameter of 167 nm and smooth surface without any beads. Furthermore, EDX spectroscopy and X-ray mapping showed uniform dispersion of GO/L-Arg-Cu in PVA NFs. The hydrophilicity of NFs was studied by measuring water contact angle (WCA) and degree of swelling. The WCA increased from 17° to 46° for the PVA/CA and PVA/GO/L-Arg-Cu/CA NFs, respectively. The antibacterial activity of the synthesized compounds was explored via Agar diffusion well method. Moreover, the inhibitory of the cross-linked PVA/GO/L-Arg-Cu NFs against bacteria causing wound infections was compared with GO, L-Arginine amino acid, CuO nanoparticles, GO/CuO nanocomposite (NC), GO/L-Arg and their nanofiber scaffolds. The PVA/GO/L-Arg-Cu/CA NFs showed the highest inhibitory zone against Escherichia coli (36 ± 0.34 mm) and Pseudomonas aeruginosa (28 ± 0.25 mm). Besides, the MTT assay on human skin fibroblast (HDF) cells related to the incorporation of copper-L-Arginine complex in nanofiber revealed a high cell viability of over 95%. These results indicated that the PVA/GO/L-Arg-Cu NFs based on an N, O-chelating may have potential application in wound healing.