The PAA (porous anodic alumina) films were prepared by two-step anodic oxidation after different times, and then the ZnO/PAA composite films were prepared by sol-gel method on their surface. Meanwhile, the ZnO/PAA composite films were characterized by X-ray diffraction (XRD), thermogravimetric/differential thermal analyzer (TG/DTA), Fourier transform infrared spectrometer (FT-IR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), selected area electron diffraction (SAED), and water contact angle (CA). The antibiofilm properties of ZnO/PAA composite films on Shewanella putrefaciens were measured simultaneously. The results show that the micromorphologies of PAA and ZnO/PAA composite films are affected by second anodization time. ZnO is a hexagonal wurtzite structure, and ZnO particles with a diameter of 10–30 nm attach to the inner or outer surfaces of PAA. After being modified by Si69, the ZnO films translate from hydrophilia to hydrophobicity. The ZnO/PAA film with the optimal antibiofilm properties is prepared on the PAA surface by two-step anodization for 40 min. The adherence of Shewanella putrefaciens is restrained by its super-hydrophobicity, and the growth of biofilm bacteria is inhibited by its abundant ZnO particles.