To study the effect of ZnO films on the formation of Shewanella putrefaciens biofilm， the ZnO films were prepared by sol-gel method and hydrothermal synthesis method on the surface of stainless steel， and the films were treated by hydrophobic modified method. The antibiofilm properties and mechanism of stainless steel and ZnO films were studied against Shewanella putrefaciens biofilm. The results show that the ZnO is hexagonal wurtzite structure， and the hydrophobic of ZnO films is stronger than that of stainless steel. The ZnO films prepared by sol-gel method are accumulated by irregularly shaped nanoparticles， the ZnO films prepared by hydrothermal synthesis method are accumulated by uniform longitudinal nanorods， and the latter is more hydrophobic. There is no difference in the stability produced by biofilm of different material surface at the same period， the major factor affecting the adhesive of biofilm is the hydrophobicity of the material surface. In the process of biofilm growth， its synthetic EPS capacity， protein content and metabolic activity are gradually enhanced， the antibacterial effect of the ZnO films prepared by hydrothermal synthesis method is significantly inhibited the metabolism and growth of the membrane bacteria， which property of inhibited biofilm is optimum.