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In this study, we extant an ecofriendly, harmless, biosynthesized zinc oxide nanoparticle by Escherichia colibacteria. Biosynthesized ZnO NPs was characterized by different techniques,and results of UV-vis showed peak at 266 nm,while X-Ray Diffractionproved the hexagonal phase structures of ZnO NPs, andFourier Transform Infrared spectra exhibited the active groups of molecules and ZnO NPswere the lower energy at 432.05. Flower shape morphology with particle size 23.87was observed in Scanning Electron Microscopeanalysis.The highest diameter of inhibition zones for P.aeroginosa, E.coli, K.pneumoniae, and A.baumanniias follows (14, 24,16, 17) mm for Ciprofloxacin and ZnO- NPs respectively. While, the diameter of highest inhibition zone for S.aureus 14mm for Tetracycline and ZnO- NPs.The effect of ZnO NPs have antibiofilm activity against MDR isolates in the presence of antibiotics (Ciprofloxacin for Gram negative and Tetracycline for Gram positive bacteria).In addition, anti-biofilm effect of ZnO NPs against A. baumannii when coated tracheal tube was studied and the result exhibit maximum inhibition of 86.7٪ at concentration 50mg ̸ ml. The effect of ZnO-NPs on WRL 68 normal cells did not show significant effect of viability rate at concentration (400, 200, 100, 50, 25). These outcomes of combined biosynthesized ZnO NPs with antibioticshowed antibacterial and antibiofilm activities against Multi-drug resistant bacteria, and when coating the medical materials. So, these nanoparticles can be further used in biomedical, pharmaceutical and other applications as an effective antimicrobial and antibiofilm agent.