This study reports the biological synthesis of gold nanoparticles (AuNPs) by the reduction of HAuCl 4 by using of Eclipta prostrata leaf extract as the reducing and stabilizing agent. AuNPs were characterized using Ultraviolet-visible (UV-vis) spectroscopy, X-ray diffraction (XRD), Fourier Transform-Infrared (FTIR) spectroscopy, Scanning Electron Microscopy (SEM), High Resolution-Transmission Electron Microscopy (HRTEM), and Energy Dispersive X-ray analysis (EDAX). The UV-visible spectrum of the synthesized AuNPs showed surface plasmon resonance (SPR) around 534 nm. The face-centered cubic (FCC) structure of the AuNPs was confirmed by XRD peaks at 38.10˝, 44.13˝, 64.43˝, and 77.32˝, which correspond to (111), (200), (220), and (311) miller indices, respectively, with clear circular spots in the selected area electron diffraction (SAED). FTIR measurements showed the AuNPs having a coating of phenolic compounds, indicating a possible role of biomolecules responsible for capping and efficient stabilization of the AuNPs. The HRTEM images determined the particles are spherical, hexagonal, and triangular in shape, with an average size of 31˘1.6 nm. The synthesized AuNPs show good antibacterial, antioxidant, and cytotoxic activity. The outcomes of this study indicate that these nanoparticles could be effectively utilized in pharmaceutical, biotechnological, and biomedical applications.