It is substantiated that nanoparticles have attracted much attention in many fields due to their extremely small size, geometry, monodispersity, non-toxicity and attractive structural and morphological features. Gold nanoparticles (Au-NPs) are a class of versatile nanomaterials used in all fields. In this study, the physicochemical analysis of Au-NPs, which are prepared from Azadirachta indica (neem leaf) extracts and aqueous chloroauric acid by biosynthesis followed by stabilization and centrifugal separation at ambient temperature, is carried out. The fabricated Au-NPs were used in the study of the antibacterial activity of Gram-negative bacteria using radial disk diffusion assay. The progress in the development of nanoparticles is monitored using ultraviolet spectroscopy, Fourier Transform Infrared, X-ray Diffraction, Energy Dispersive X-ray analysis and scanning electron microscopy. The characterization results confirm the successful synthesis of nanoparticles with high yield and well-structured surface morphology and elemental composition, which is confirmed by Scanning Electron Microscopy Image and Energy Dispersive X-ray spectra.