This publication is part of a Special Collection for the Conference on ''Cutting-Edge Research in Materials and Sustainable Chemical Technologies'' (CRMSCT-2022). Please follow to visit the whole collection.Four bacterial strains were used for biosynthesis of zinc oxide (ZnO) nanoparticles (NPs), namely Bacillus tropicus SHJP5, Bacillus tropicus LKJP6, Aeribacillus composti SJP25, and Aeribacillus pallidus SJP28. The synthesized nanoparticles were characterized using UV-Visible spectroscopy (UV-Vis), Fourier transform infrared spectroscopy (FTIR), Field Emission Scanning Electron Microscopy (FESEM), Photoluminescence spectroscopy (PL), Thermogravimetric analysis (TGA), and X-ray diffraction (XRD). The XRD pattern of NPs (bio-synthesized) shows the polycrystalline hexagonal wurtzite structure with the three major planes (100), (002), and (001). The average crystallite size determined by the Debye-Scherrer equation is between 8-54 nm. A reduction in crystallite size of as-prepared nanoparticles was observed in the presence of bacterial strains. The FTIR analysis reveals the amide, carboxyl, and hydroxyl groups found on the surface of biosynthesized ZnO NPs. These groups behave as reducing and stabilizing agents. The UV-Vis spectra reveal the absorption bands for control, SHJP5, LKJP6, SJP25, and SJP28 at 373, 363, 382, 358, and 380 nm respectively. The corresponding energy band gap was found to be 3. 324, 3.415, 3.246, 3.463, and 3.263 eV respectively, with the decreasing size of the nanoparticles produced by bacterial strains. The FESEM analysis confirms the hexagonal structure of bio-synthesized ZnO NPs. The nanoparticles have a uniform distribution of spherical shape except for the strain Aeribacillus pallidus LKJP6 which exhibits uniform and thin nanorods. Minimum inhibitory concentration (MIC) tests against E. coli (7.1 mg/ml) and S. aureus (3.2 mg/ml) verified the antibacterial activity of produced ZnO NPs. The use of isolated strains for the extracellular manufacture of ZnO NPs is encouraged in this work. To the best of our knowledge, this might be the first study that has been presented that shows the structural, optical, and surface morphology of a thermophilic bacterium, Bacillus tropicus SHJP5, Bacillus tropicus LKJP6, Aeribacillus composti SJP25, and Aeribacillus pallidus SJP28 for the biosynthesis of ZnO NPs in particular.