Background and Objectives: The advantages of biological methods are Low cost, non-toxicity, production of high purity nanoparticles, low time-consuming, and completeness reation time. In the present study, biosynthesis of gold nanoparticles using aqueous extract of Seidlitzia rosmarinus and thier antimicrobial activity, were investigated. Methods: In this study, after preparing the extract, 2 ml of it was added to 4 ml of HAuCl4.3H2O with concentration of 1mM, and the color of solution immediately changed to purple. The parameters affecting the synthesis of nanoparticles [such as pH of reaction medium, extract volume, concentration of gold (III) salt, temperature, and reaction time], were assessed and optimized using UV-vis spectrophotometry. A transmission electron microscope (TEM) was used to characterize the nanoparticles. The antibacterial activity of nanoparticles against four species of pathogenic bacteria (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Salmonella typhimurium), were assessed by agar well diffusion method. Results: Gold nanoparticles showed the maximum absorbance at 544 nm. It was found that the nanoparticles have spherical shape and their average size was 10-18 nm. Also, the results of antibacterial activities showed that the synthesizied nanoparticles had relatively high antibacterial activity. Conclusion: In the current study, the aqueous extract of Seidlitzia rosmarinus due to its seccondary components and antioxidant activity, has high potential in reduction of gold metal ions as well as synthesis and stabilization of gold nanoparticles, and these synthesized gold nanoparticles were also relatively high antibactrial activity.