Zinc oxide (ZnO) films were prepared on aluminum substrate by a hydrothermal method to investigate the effect of their surface characteristics, including morphology and hydrophobicity, on the corresponding antibiofilm performance. The surface characteristics of the prepared ZnO films were examined by a comprehensive range of methodologies, suggesting that films of distinctive surface morphologies were successfully formed. Subsequently, their antibiofilm activities, using Shewanella putrefaciens as a model bacterium, were assessed. Surface measurements confirmed that the ZnO films equipped with a nanoscopic needlelike surface feature are more hydrophobic than those possessing densely packed microflakes. The reduced number of live cells and presence of biofilm, confirmed by optical and electron microscopy results, suggest that the former films possess an excellent antibiofilm performance. It is believed that the engineered nanoscopic needle feature might penetrate the cell membrane when they are in contact, allowing the effective substance of ZnO antibacterial ingredients to diffuse into the embedded bacteria. Furthermore, such surface characteristics might perturb the integrity of the cell membrane causing the intracellular substance is leaked from the cells. As such, the combinatorial effects of nanoscopic feature resulted in an inhibited growth of S. putrefaciens biofilm on ZnO film.