Polyurethane (PU) nanofibers containing graphene oxide (GO) and Ag doped functionalized reduced graphene oxide (Ag-RGO) were successfully prepared via the electrospinning technique. The uniform distribution of GO sheets along with Ag nanoparticle in the nanofibers was investigated by scanning electron microscopy and the elemental mapping technique. X-ray diffraction and thermal gravimetric analysis verified the presence of GO and Ag in the bicomposite nanofibrous mats. Antibacterial tests against Escherichia coli demonstrated that the addition of GO and Ag-RGO to the PU nanofiber greatly enhanced bactericidal efficiency. Overall, these features of the synthesized nanofibers make them a promising candidate material in the biomedical field for applications such as tissue engineering, wound healing, and drug delivery systems.