Metal and semiconductor nanoparticles synthesis is an growing field of interest, owing to possible applications in the development of new technologies. Biologically synthesized nanomaterial has recently evolved into an significant branch of nanotechnology. The present work identified the synthesis of Zinc Oxide Nanoparticles (ZnO NPs) using Acacia farnesiana (L.) leaf aqueous extract, antifungal and its antimicrobial activities. The nanoparticles were characterized by the study of UV- visible spectroscopy, Scanning electron microscope (SEM), and X-ray diffraction (XRD). Furthermore, ZnO NPs showed significant bactericidal effect against both Gram+ve and Gram-ve bacterial pathogen strains in an agar well diffusion method. These findings emphasize that such biocompatible ZnONPs with multifunctional activities may find their application in the field of nanomedicine.