The aim of this study is to green-synthesize gold nanoparticles (GNPs) by leaf infusion of Achillea wilhelmsii (AW) and to evaluate their biological effects. The synthesized AW-GNPs were characterized by UV–Vis spectroscopy, Fourier transform infrared, X-ray diffraction (XRD), transmission electron microscopy (TEM) and dynamic light scattering analyses. The antibacterial and cytotoxicity activities of AW infusion and AW-GNPs were evaluated. Antioxidant activity was evaluated by 1, 1-diphenyl-2-picrylhydrazyl test. Moreover, the electrochemical activity of AW-GNPs as a modifier ata glassy carbon electrode (GCE) was studied. The surface plasmon resonance absorption band at 540nm in the UV–Vis spectrum discloses the reduction of gold cations into GNPs. The XRD pattern of GNPs shows the crystal structure of AW-GNPs, and TEM image displays the spherical shape of GNPs with small size (2.7–38.7 nm). The AWinfusion and AW-GNPs demonstrated a maximum antioxidant activity of 68 and 58%, respectively. AW-GNPs showed antibacterial activity against gram-positive bacteria, without affecting gram-negative bacteria. Also, AW-GNPs exhibited the cytotoxicity effects against MDA-MB-468 cancer cell line, and the electrochemical study indicated a significant electrocatalytic activity of AW-GNPs. The results indicate the advantages of using AW leaf infusion for the production of GNPs with antibacterial, antioxidant, cytotoxic and electrocatalytic activities.