ABSTRACT The synthesis of nanostructured materials has gained significant interest due to their unique properties and applications in various fields. The biological approach of nanoparticle synthesis is cheaper and environmentally friendly compared to conventional chemical methods. This study was aimed to synthesize Zinc oxide nanoparticles (ZnO NPs) using actinomycetes and evaluate their antimicrobial properties. The synthesized nanoparticles were characterized using various techniques (UV-Vis spectroscopy, Fourier transform-infrared spectroscopy, scanning electron microscopy, X-ray diffraction analysis, and Zeta potential analysis) . Their antibacterial activity was tested using the well diffusion method against four different bacteria species. The transmission electron microscope showed that the average size of ZnO NPs was 20–38 nm, while X-ray diffraction and energy-dispersive X-ray spectroscopy confirmed the purity of the biosynthesized ZnO NPs. The biosynthesized ZnO NPs showed effective antibacterial activity against E. coli MTCC 739, Pseudomonas aeruginosa MTCC 2453, Staphylococcus aureus MTCC 96, and Bacillus subtilis MTCC 736. Thus, biosynthesized ZnO NPs have the potential to be stable and eco-friendly nanoparticles with antibacterial activities.