In the presented study, characterisation and synthesis of zinc oxide NPs (ZnONPs) through Bacillus subtilis (B. subtilis) are studied. To analyze the formation of ZnONPs, B. subtilis has been cultured in Nutrient broth as well as incubated at a temperature of 37 Celsius for a period of 24 hrs. The creation of ZnONPs was demonstrated when the supernatant following centrifugation was introduced to 1 mM ZnO and the color changed from yellow to light yellow. The structure, morphology and stability of the created ZnONPs have been examined with the use of morphological and structural properties technologies as FE-SEM analysis and XRD. Additionally, optical properties, like UV and FTIR spectra have been used. The results showed that the created NPs have been largely stable, hexagonal in phase, roughly spherical, with maximum particles with a diameter between 7 and 19 nm. The antibacterial effectiveness of ZnONPs against several dangerous microorganisms was also examined in this study. According to the research, ZnONPs have both a fatal and higher suppressive effect on pathogenic bacteria.