In this study, ZnONPs were biosynthesized from aqueous zinc sulphate solution through a simple and ecofriendly method using Pseudomonas aeruginosa culture filtrate, which acted as a reductant and stabilizer simultaneously. ZnONPs were characterized using UV-Vis spectroscopy, XRD, FTIR, SEM, AMF, and zeta-potential analysis.The surface plasmon resonance peaks in absorption spectra for ZnONPs colloidal solution showed an absorption peak at 378 nm in a UV-visible spectrum. The functional biomolecules such as carboxyl groups present in the Pseudomonas aeruginosa culture filtrateresponsible for the zinc oxide nanoparticles formation were characterized by FT-IR. The results of AFM show the tip-corrected measured the size of ZnONPs in the range of 30-80 nm. And the 3D view of the sample surface and height distribution around 50 nm.The XRD results suggested that the crystallization of the bio-organic phase occurs on the surface of the ZnONPs or vice versa. The broadening of peaks in the XRD patterns was attributed to particle size effects and the average particle size of ZnONPs can be calculated using the Debye -Scherrer equation, it was found about 20-40 nm. The ZnONPs were scanned using SEM. It reveals that a ZnONPs seems to be irregular and polymorphic where the crystalline forms of these particles varied between rectangular, spherical, radial and cylindrical structures and the average size ranges between 28-43 nm. Measuring the stability of the colloidal solution of ZnONPs gave a value of -51.8 Mv.The result shows the ability of ZnONPs to inhibit the growth and the biofilms of Pseudomonas aeruginosa isolates.We concluded the biosynthesized ZnO NPs which have antibacterial and antibiofilm activities and it can be used as an antibacterial therapeutic agent.