The purpose of this study was to evaluate the effect of nano-zinc oxide (ZnO-N) morphology on the functional and antimicrobial properties of tapioca starch films. For this reason, nanosphere (ZnO-ns), nanorod (ZnO-nr), and nanoparticle of ZnO (ZnO-np) at 0.5%, 1.0%, and 2.0% were added to the starch film. Then, physicochemical, mechanical, and barrier properties were evaluated. Also, UV-visible and Fourier transform infrared spectroscopy (FTIR) spectra and antibacterial activity of prepared nanocomposite films against Escherichia coli were examined. The results revealed that the ZnO-ns had the most effects on mechanical, physicochemical, and barrier properties. The highest values of the tensile strength (14.15 MPa) and Young's modulus (32.74 MPa) and the lowest values of elongation at break (10.40%) were obtained in the films containing 2% of ZnO nanosphere. In terms of UV transmission, ZnO-nr showed the most significant impact morphology. FTIR spectra indicated that interactions for all morphologies were physical interaction, and there are no chemical reactions between starch structure and nanoparticles. The antibacterial effect of the ZnO-ns was higher than that of other morphologies. In summary, ZnO-ns was the best morphology for using ZnO-N in starch-based nanocomposite films.