In this paper, the synthesis of colloidal solution of ZnO nanoparticles by PLAL using an Nd: YAG laser with 1064 nm and 532 nm wavelengths is described. The optical, morphological, structural, wettability and antibacterial activities were studied. The XRD analysis showed the hexagonal wurtzite structure while the UV-vis indicated the blue shift of the absorption peak due to the surface plasmon of the nanoparticle, which showed significant changes in color behavior. The FE-SEM revealed a uniformed spherical shape with a partial size range of 20.50–21.48 nm for ZnO at 1064 and 57.08–59.87 nm for ZnO at 532nm. Also, the concentration measurement indicated that the concentration of ZnO prepared at 1064 nm is 26.7 μg ml-1 and 63 μg ml-1 at 532 nm due to the relevance of high energy to the Zn plate and little distortion in the water and high absorption of short wavelength. Finally, the optical density of the antibacterial activity has been investigated, and both concentrations have good antibacterial activity at 12 h. The ZnO prepared at 532 nm has higher antibacterial activity because it has a high concentration of nanoparticles for both S. aureus and E. coli. Finally, the cell viability for ZnO synthesis with both 1064 nm and 532 nm indicates that this material has high biocompatibility and could be used for medical applications.