The Colloidal solutions of nickel oxide (NiO) nanoparticles synthesized via Nd-Yag pulse ablation of nickel immersed in H2O were studied. The created nanoparticles were characterized by UV-VIS absorption, Fourier transform infrared spectroscopy (FTIR) and transmission electron microscope (TEM). FTIR characterization confirms the formation of nickel oxide nanoparticles. The optical band gap values, determined by UV-VIS absorption measurements, are found to be (4.5 ev). TEM shows that nanoparticles size ranged from 2-21 nm. The antimicrobial activity was carried out against pseudomonas aurogenisa, Escherichia coli (gram negative bacteria), Staphylococcus aureus and Streptococcus pneumonia (gram positive bacteria). The NiO nanoparticles showed inhibitory activity in both strains of bacteria with best selectivity against gram-positive bacteria. The findings of present study indicate that NiO nanoparticles could potentiate the permeability of bacterial cell wall, and remarkably increase the accumulation of amoxicillin in bacteria, suggesting that NiO nanoparticles together with amoxicillin would facilitate the synergistic impact on growth inhibition of bacterial strains.