OBJECTIVE The aim of the present study was preparation, physicochemical characterization and performance evaluation of gold nanoparticles (GNPs) in radiotherapy. Another objective was the investigation of anti-bacterial efficacy of gold nanoparticle against E. coli clinical strains. METHODS Gold nanoparticles prepared by controlled reduction of an aqueous HAuCl4 solution using Tri sodium citrate. Particle size analysis and Transmission electron microscopy were used for physicochemical characterization. Polymer gel dosimetry was used for evaluation of the enhancement of absorbed dose. Diffusion method in agar media was used for investigation of anti-bacterial effect. RESULTS Gold nanoparticles synthesized in size range from 57 nm to 346 nm by planning different formulation. Gold nanoparticle in 57 nm size increased radiation dose effectiveness with the magnitude of about 21 %. At the concentration of 400 ppm, Nano gold exhibited significant anti-bacterial effect against E. coli clinical strains. CONCLUSIONS It is concluded that gold nanoparticles can be applied as dose enhancer in radiotherapy. The Investigation of anti-bacterial efficacy showed that gold nanoparticle had significant effect against E. coli clinical strains.