Biosynthesized nanoparticles have an incredible application in biomedicine owing to its simplicity, eco-friendly properties and low cost. The present study aims to determine the green synthesized zinc oxide nanoparticles from methanolic leaf extract of Glycosmis pentaphylla. The synthesized nanoparticles were characterized using UV-VIS Spectroscopy, Flourescence spectrometer, FT-IR, XRD, SEM with EDAX and TEM. The confirmations of synthesized nanoparticles were characterized by peak at 351 and 410 nm in the UV-VIS spectrum and photoluminescence spectrum respectively. FT-IR studies revealed the functional group of the nanoparticles. The XRD data showed the crystalline nature of the nanoparticles and EDAX measurements indicated the 20.70% of highly pure zinc oxide metal. The morphological characterization of synthesized zinc oxide nanoparticles was analyzed by SEM and TEM and size of the particles were ranging from 32 to 36 nm. The synthesized zinc oxide nanoparticles exhibited interesting antimicrobial activity against pathogenic organisms. In addition, this is the first report on leaf mediated synthesis of zinc oxide (ZnO) nanoparticles from Glycosmis pentaphylla.