Bio-templates provide a facile, rapid, and environmentally benign route for synthesizing various nanostructured materials. Herein, Locust Bean Gum (LBG), a galactomannan polysaccharide, has been used as a biotemplate for synthesizing ZnO nanoparticles (NPs) for the first time. The composition, structure, morphology, and bandgap of ZnO were investigated by Energy Dispersive X-ray Spectroscopy (EDX), X-Ray Photoelectron Spectroscopy (XPS), X-ray powder diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and UV-vis spectroscopy. XRD data showed single-phase crystalline hexagonal NPs. FTIR spectra confirmed the presence of M-O bonding in the sample. At a concentration of 0.5 mg/mL the NPs can degrade Rhodamine B under sunlight, displaying excellent photocatalytic activity. These NPs exhibited antimicrobial activity in both Staphylococcus aureus and Bacillus subtilis. Significant cell death was observed at 500 μg/mL, 250 μg/mL, 125 μg/mL and 62.5 μg/mL of NP in breast cancer, ovarian cancer and lung cancer cell lines. Wound healing assay showed that the NPs significantly blocked the cell migration at a concentration as low as 62.5 μg/mL in all three cell lines. Further optimization of the nanostructure properties will make it a promising candidate in the field of nano-biotechnology and bioengineering owing to its wide range of potential applications.Copyright © 2023. Published by Elsevier B.V.