In this work, zinc oxide nanoparticles (ZnO - NPs) were prepared using a sol-gel methodology and tested for their antibacterial activity against each of the following pathogenic species: Escherichia coli, Klebsiella pneumonia, and Staphylococcus aureus by well diffusion assay. The sample prepared was characterized by different techniques: Atomic Force Microscope AFM; Fourier Transform Infrared FT-IR; Scanning Electron Microscope SEM and X-Ray Diffraction Spectroscopy XRD. The XRD result showed that ZnO - NPs presence in wurtzite the structure of ZnO. The AFM and SEM of the surface analysis indicate that the most ZnO – NPs appear approximately in a spherical shape with some agglomeration. The average particle size for ZnO - NPs is nearly 37 nm. Volumes 25µl, 50µl, 75µl, 100µl, 125µl, and 150µl of 10 mg\ ml concentration of ZnO - NPs were used, the antimicrobial activity results showed that ability for ZnO - NPs to inhibit the growth of S.aureus increased as the solution volume increased, while the growing of (K. pneumonia) and (E. coli) was inhibited only with the volume 75µl where the inhibition zones diameters were 15mm and 10mm respectively.