Modified polyurethane (PU) nanofibers were prepared by an industrial electrospinning thechnique Nanospider. Copper oxide (CuO), known for its antibacterial properties, was selected as a modifier. A simple method of incorporation of CuO microparticles in different concentrations into a polymer solution has utilized to prepare PU/CuO nanocomposites. The aim of present research was to study an influence of different amounts of CuO on the rheological properties of PU solution and on the structure and dimensional characteristics of the prepared modified nanolayers. Viscosity and conductivity of polyurethane solutions were studied using Rheometer HAAKE Roto Visco 1 and conductivity meter EUTECH instruments con 150 respectively. The structure of obtained samples and the content of copper were investigated by SEM-EDX analyze. The average diameters, fiber uniformity coefficients and surface densities of pristine and modified samples were calculated and analyzed. Microorganisms Escherichia colt and Staphylococcus aureus had been used to prove the antimicrobial properties of nanofiber's mats. The interesting conclusion of the present study is a positive impact of CuO on the spinning performance of PU nanofibers. Selected modifier has shown itself as an additive which increases the performance, has no significant effect on the dimensional characteristics of produced polyurethane mats and imparts them antibacterial properties.