Recently, zinc oxide nanoparticles (ZnO NPs) have been used in several biological and industrial applications due to their low toxicity, high efficiency, biocompatibility, and cost-effectiveness. The present study used Bacillus subtilis ATCC 6633 in the biosynthesis of the antimicrobial ZnO NPs. The synthesized ZnO NPs were characterized using UV-Visible spectroscopy, Fourier transform-infrared (FTIR) spectroscopy, X-ray diffraction (XRD) spectroscopy, Zeta potential analysis, and transmission electron microscopy (TEM). The TEM results confirmed the spherical shape of ZnO NPs with a size range of 13.5-30.4 nm. Zeta potential analysis showed the positive charge of ZnO NPs (+16.8 mV). The ZnO NPs showed good antimicrobial activity against the tested pathogenic bacterial strains; B. cereus (Gram-positive bacteria) in comparison to Escherichia coli (Gram-negative bacteria). They also revealed a high antifungal activity against the tested pathogenic fungal strain; Aspergillus niger. Here, this cost-effective and simple biological approach for ZnO NPs synthesis has emerged with a promising capacity in biomedicine, especially in the fields of antibacterial and antifungal fields.