Biological sources of microorganisms and plants are playing a major role in the reduction of metallic nanoparticles such as silver and gold, as it emerges as an eco-friendly and exciting approach in nanotechnology. We report on the biological synthesis of gold nanoparticles using the culture supernatant of Bacillus subtilis and its effect on increased antibacterial and antifungal activities against clinically isolated organism. When the supernatant of Bacillus subtilis was added to HAuCl4 aqueous solution, HAuCl4 was reduced as Au+ ions, which confirmed the presence of nanoparticles by the color change of pale yellow to purple. The minimum and maximum peaks were observed at 24th and 120th hours by UV-Visible spectroscopy. The combined antibacterial and antifungal activities with various antibiotics were observed against clinical isolates.