We report on the preparation and characterization of polyurethane (PU) nanofibers containing silver (Ag) nanoparticles were synthesized by using electrospinning. Two different approaches were adopted to incorporate the Ag nanoparticles in to PU nanofibers. In the first approach, a homogeneous solution of 10 wt% PU containing silver nitrate was electrospun to obtain PU-Ag composite nanofibers. And in the second approach, the pristine PU nanofibers were initially electrospun and then Ag nanoparticles were coated via wet casting method. The surface morphology, structure, bonding configuration, optical and thermal properties of the resultant products were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, UV-vis spectroscopy and thermogravimetric analysis. The antibacterial activity was tested against four common food borne pathogenic bacteria, namely, Staphylococcus aureus, Escherichia coli, Salmonella typhimurium and Klebsiella pneumoniae by minimum inhibitory concentration (MIC) method. Our results demonstrated that no bactericidal activity was detected for the pristine PU nanofibers. Further on, antibacterial activity was observed to be more pronounced for the composite nanofibers which were attributed to the presence of Ag nanoparticles in the composite nanofibers. Overall, this study demonstrates the fabrication of cheap, stable and effective nanofiber mats with excellent antimicrobial activity that can be utilized to inhibit the microbial growth associated with food stuff.