The effective treatment of cancer and bacterial pathogens are two key challenges in modern nanomedicine. Here, zinc oxide nanoparticles (ZnO NPs) were fabricated using the crustacean immune molecule β-1, 3- glucan binding protein (Phβ-GBP, 100kDa) purified from the heamolymph of Paratelphusa hydrodromus. β-GBP coated zinc oxide nanoparticles (Phβ-GBP-ZnO NPs) were characterized by UV-vis spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and high resolution-transmission electron microscopy (HR-TEM) analyses. Phβ-GBP-ZnO NPs inhibited the growth of Staphylococcus aureus and Proteus vulgaris. Protein and nucleic acid leakage assays showed that Phβ-GBP-ZnO NPs facilitate membrane permeability leading to cell death. The antibacterial activity of Phβ-GBP-ZnO NPs was due to the high level of reactive oxygen species (ROS) release from bacterial cells post-treatment with 75μg/mL of Phβ-GBP-ZnO NPs. Confocal laser scanning microscopy pointed out that biofilm thickness was highly reduced post-treatment with nanoparticles. Cytotoxicity on human liver carcinoma (HepG2) cells highlighted that 75μg/mL of Phβ-GBP-ZnO NPs inhibited viability of HepG2 cells. Phase contrast microscopy showed key morphological changes of HepG2 cells post-treatment with Phβ-GBP-ZnO NPs. Overall, Phβ-GBP-ZnO NPs can be further considered for the development of novel drugs against microbial pathogens and HepG2 cells.