An essential component of nano-biotechnology is the production of reliable and eco-friendly routes for synthesizing nanoparticles. Plant extracts are an affordable, cost-effective and eco-friendly material for the synthesis of nanoparticles. In the current study, gold nanoparticles (AuNPs) were bio-synthesized utilizing E. globulus leaf extract. Rapid reduction of the gold ions into AuNPs is seen once the gold ions are exposed to an aqueous solution of leaf extract. TEM, EDX and UV-Vis spectroscopy methods were used to investigate these bio-synthesised AuNPs. The aqueous media containing AuNPs had a peak in the UV-visible spectrum at 540 nm. According to TEM investigation, AuNPs were evenly dispersed and had spheres with spherical and triangular forms that were between 15 and 30 nm in size. By using the agar well diffusion method, the antibacterial activity of the biosynthesized AuNPs was confirmed against prevalent human pathogens such S. aureus (NCIM 5021) and P. aeruginosa (NCIM 5029). This process uses cheap, easily accessible biomaterial to synthesize metallic nanoparticles.