In this study, green synthesis, characterization and antibacterial activity of zinc oxide nanoparticles was studied. The zinc oxide nanoparticles were successfully synthesized via a fast, convenient, cost effective, and environmentally friendly method by biologically reducing Zn (NO 3 solution with Neem (Azadirachta indica) leaf extract under optimum condition. The presence of active flavonoids, phenolic groups, alkaloids, terpenoids and tannins, which were in the biomass of the Neem leaf extract before and after reduction was identified using qualitative screening methods (observing the colour changes) and FT-IR Spectroscopy. The formation of ZnO NPs was visually indicated by the color changes from colorless to light yellow color. Biosynthesized nanoparticles were also characterized by UV-Visible, FT-IR, and XRD spectroscopies. The reduction process was simple and convenient to handle and was monitored by UV-Visible spectroscopy that showed surface plasmon resonance (SPR) of the ZnO NPs at 321 nm. This result clearly revealed the formation of ZnO NPs. X-ray diffraction was used to investigate the crystal structure. The average particle size of ZnO powder and around 20 nm using the line width of the plane, refraction peak using the Scherrer’s equation. Green synthesized zinc oxide nanoparticles were evaluated for antimicrobial activities against Gram positive and Gram negative bacteria. Zinc nanoparticles exhibited maximum zone of inhibition against Escherichia coli (15 mm) while least activity was seen against Staphylococcus aureus. Keywords: ZnO NPs, Green synthesis, Neem leaf, UV-Vis, FT-IR, XRD, Antibacterial activity ) 2