Fifty specimens were collected from wound patients who visited Al-Hilla Teaching Hospital. The samples were grown on Blood and MacConkey agar for 24-48 hr at 37oC. The bacterial isolates which achieved as a pure and predominant growth from clinical samples as Pseudomonas fluorescens, were identified using morphological properties and Vitek2 system. The anti-bacterial activity of copper oxide nanoparticles (CuO NPs) against was tested by (disk diffusion assay) using dilutions of (400, 200, 100, 50, 25, and 12.5‎µ‎g/ml). The (MIC and MBC) of each isolate was determined. CuO NPs shows wide spectrum antibacterial activity against tested bacteria with rise zone of inhibition diameter that is proportionate with the increase in nanoparticle concentration. The MIC of CuO NPs extended from 100-200‎µ‎g/ml and the MBC ranged from 200-400‎µ‎g/ml. The antibiotic profile was determined by Viteck 2 compact system (Biomérieux). CuO NPs‎ found highly effective and safe in P. fluorescens wounds infections comparing with used antibiotics.