Half-fin anchovy(Setipinna taty) hydrolysates (HAHp) was conjugated with zinc oxide nanoparticles (ZnO NPs) using a hydrothermal method to develop a novel antibacterial nanocomposite. The generated supernatants of the conjugate, designated as HAHp(3.0)/ZnO NPs, were characterized by transmission electron microscopy, high resolution transmission electron microscopy, and inductively coupled plasma-optical emission spectrometer. Results showed that HAHp(3.0) was absorbed on the surface of the ZnO NPs. The total content of zinc element was 9127.4 mg/kg in HAHp(3.0)/ZnO NPs. The increased antibacterial effects were observed for the HAHp (3.0)/ZnO NPs with the minimal inhibitory concentration of 3.5 mu gprotein/mL against Escherichia coif (E. colt), Pseudomonas fiuorescens, Salmonella and Staphylococcus aureus, compared to the bare HAHp(3.0). The antibacterial activity of HAHp(3.0)/ZnO NPs was further evaluated using E. colt as the model strain. The incubation of HAHp(3.0)/ZnO NPs increased the outer and inner membrane permeability in E. coif cells, and the leakages of potassium ions and the cytoplasmic beta-galactosidase were detected during the process. Furthermore, porous structures were observed on the membrane of E. coil cells by scanning electron microscopy. In addition, the formation of intracellular reactive oxygen species was detected using fluorescence microscopy. The results suggested that the HAHp(3.0)/ZnO NPs could be a promising antibacterial nanocomposite.