THE SPREAD of resistant bacteria and the development of bacterial biofilm are two major challenges in the application of biomaterials. The overuse of antibiotics has become a common cause of the emergence of multidrug-resistant (MDR) bacteria. Besides, biofilm infections are notoriously difficult to treat, as the biofilm matrix provides physical protection from antibiotic treatment. Therefore, a variety of new antimicrobial drugs has attracted wide attention in treating infectious diseases developing from MDR bacteria and bacterial biofilms. These drugs are related to an important group based on the use of nanoparticle-based materials. Metal oxide nanoparticles including zinc peroxide nanoparticles (ZnO2-NPs) exhibit remarkable antimicrobial activities against MDR bacteria and hence are one of the most propitious alternative antimicrobial agents. Herein, the antibacterial activity of the synthesized ZnO2-NPs was investigated against 7 clinical MDR Pseudomonas aeruginosa strains using disc diffusion assays on Muller-Hinton agar. These strains were also multi-virulence producers with special reference to hemolysin, pyocyanin, gelatinase, protease, lipase and biofilm production. Clearly, the remarkable significant bactericidal activity of ZnO2-NPs against tested strains was exhibited, with a maximum zone of inhibition of 19.81±1.5mm against P. aeruginosa strain 22 (PA-22) at a concentration of 300μg/ml. In addition, ZnO2-NPs exhibited significant anti-biofilm activity by inhibiting bacterial biofilm formation as revealed spectrophotometrically. This study established the possibility of developing antimicrobial ZnO2-NPs to combat developing drug resistance and biofilm-related infections.