Green synthesis is a simple, low-cost, non-toxic, environmentally friendly and efficient approach to use. Leaf extract of plants rich in polyphenols, such as flavonoids, is a powerful agent in reducing the synthesis of gold nanoparticles. The purpose of this study is to investigate the parameters affecting the biosynthesis of gold nanoparticles using the aqueous extract of Scrophularia striata plant and their antimicrobial activity. Biosynthesis of gold nanoparticles was accomplished by the interaction of golden salt (HAuCl4, 3H(2)O) with aqueous extract of Scrophularia striata. In order to obtain uniform and spherical nanoparticles, the following parameters affecting the biosynthesis of nanoparticles were investigated and optimized by ultraviolet-spectrophotometric technique; golden salt concentration, extract volume, pH and reaction time. Transmission electron microscopy and X-ray diffraction technique were also used to further characterize nanoparticles. Finally, the anti-bacterial properties of gold nanoparticles were investigated by disc diffusion method. The resulting absorption spectra exhibited strong peaks at 570 nm, which is a specific wavelength for gold nanoparticles. Transmission electron microscopy studies showed that the gold nanoparticles had a spherical shape with a mean diameter of 5-10nm, and the highest diameter of the growth inhibition zone was observed on the diameter of the hafnium bacteria (14mm). In this study, it was observed that, with the aid of Scrophularia striata aqueous extracts, a golden nanoparticle showed an antibacterial activity against gram-negative bacteria.