In nanotechnology, using of metallic nanoparticles (MNPs) has obtained major attention for over a century because of their unique properties in nano scale. In this paper, the effect of three factors (incubation time, concentrations of copper sulfate and glucose) on the biosynthesis of copper (Ⅱ) oxide nanoparticles (CuO NPs) by Halomonas elongata IBRC-M 10214 and the antibacterial activity of nanoparticles were evaluated. Experiment design and analysis of Taguchi method were carried out by Qualitek-4 software. Effect of CuSO4 concentration (1.4, 2.8 and 5.6 mM), incubation time (48, 72 and 96 h) as three different levels was measured as three major factors in the biosynthesis of metallic nanoparticles. Synthesized CuO nanoparticles were characterized by utilizing ultraviolet-visible (UV-Vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infra-red (FT-IR) spectroscopy and field emission scanning electron microscope (FESEM) techniques. Among the three factors, results illustrated that considerable effect was related to CuSO4 concentration. These analyses demonstrated that the average CuO nanoparticles crystalline size was 57-79 nm with rectangular shape. Also, for evaluating of antibacterial effects, maximum zone of inhibition, two important multidrug resistant pathogenesis bacteria, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 43300 were used. Antibacterial assay of CuO nanoparticles showed antibacterial activity toward the pathogenic bacterial strains of E. coli by 5 mm and S. aureus by 5.5 mm for maximum zone of inhibition. In conclusion, this study presented simple, low expensive, eco-friendly and high productivity in the fabrication of CuO nanoparticles. In addition, these metallic nanoparticles had antibacterial effect that could be usable in medicinal aspect for fighting against prominent pathogen bacteria such as E. coli ATCC 25922 and S. aureus ATCC 43300.