Nanotechnology is providing new ways to manipulate the structure and chemistry of surfaces to inhibit bacterial colonization. In this study, we evaluated the ability of glass slides coated with zinc oxide (ZnO) nanoparticles to restrict the biofilm formation of common bacterial pathogens. The generation of hydroxyl radicals, originating from the coated surface, was found to play a key role in antibiofilm activity. Furthermore, we evaluated the ability of the nanoparticle coating to enhance the antibacterial activity of commonly-used antibiotics. The ZnO nanoparticles were synthesized and deposited on the surface of glass slides using a one-step ultrasound irradiation process. Several physico-chemical surface characterization methods were performed to prove the long-term stability and homogenity of the coated films. Collectively, our findings may open a new door for utilizing ZnO nanoparticle films as antibiofilm coating of surfaces, thus providing a versatile platform for a wide range of applications both in medical and industrial settings, all of which are prone to bacterial colonization.