Green nanoparticle synthesis is gaining popularity and has been proposed instead of using physical and chemical processes. In this work, the bacterial strain Stenotrophomonas pavanii was used to produce zinc oxide nanoparticles (ZnO-NPs) in a low-cost and eco-friendly manner. The biosynthesized nanoparticles were examined using dynamic light scattering (DLS), X-ray diffraction (XRD) and transmission electron microscopy (TEM). The ZnO nanoparticles synthesized were clean, principally round in form, and vary in size from 0.9 to 17.1 nm. In the present work, the biosynthesized ZnO nanoparticles have been used for antimicrobial applications against Gram-positive and Gram-negative bacteria, yeast, and fungi strains. ZnO NPs had better antibacterial efficacy against tested Gram-negative bacteria than they did against Gram-positive bacterial and fungal strains. The highest antimicrobial activity against Escherichia coli (28 mm) and Pseudomonas putida (29 mm). The extracellular production of zinc oxide nanoparticles from culture Stenotrophomonas pavanii showed good antibacterial activity.