This study designed to investigate the properties of antibacterial nanofiber scaffolds of polyurethane-Cinnamomum zeylanicumagainst virulence gene expression inhibition ofPseudomonas aeruginosaandStaphylococcus aureusthat are important in burn wounds. With attention to burn wound infections in hospitals and mortality increase in patients, it is necessary to design nanodressing. Clinical isolates were confirmed by biochemical and microbiological tests. DNA of isolates was extracted and PCR used to confirm thealpgene ofP. aeruginosaandPvgene ofS. aureus. Polyurethane nanofiber and cinnamon polymers were used to prepare the scaffold under the electrospinning process. Infrared spectroscopy, electron microscopy, and mechanical tensile tests were used to confirm the scaffolds. The susceptibility testing and minimum inhibitory concentration of polyurethane-cinnamon nanofiber scaffold were determined againstP. aeruginosaandS. aureus. For confirmation of polyurethane-cinnamon nanofiber scaffold were used the cytotoxicity test (MTT), FTIR, mechanical tensile test, and a scanning electron microscope. The expression of virulence genes was investigated using the real-time RT-PCR technique. The results of the susceptibility testing indicated thatP. aeruginosaandS. aureuswere susceptible to polyurethane-cinnamon nanofiber scaffold. The MTT, FTIR, mechanical tensile test, and SEM confirmed the different features of the polyurethane-cinnamon nanofiber scaffold. Results of real-time PCR demonstrated that the expression levels ofp-vandalpgenes after treatment decreased, respectively, 2.71- and 1.06-fold. Results indicated that the electrospun polyurethane-cinnamon nanofiber scaffold for the first time could inhibit both important pathogens of the hospital and the expression of the virulence genes. Considering the susceptibility ofP. aeruginosaandS. aureusto and its inhibitory effect onan alpandp-vgenes, this system could probably be a candidate in wound dressing for commercial purposes to burn healing and infection inhibition.