This study aims to introduce a green, eco-friendly, fast and cost effective method to synthesis gold nano-sized particles (Au NPs) from actinomycete strain; Streptomyces sp. U30 (KP109810), isolated from metal containing rock. The biosynthesized gold nanoparticles were formed at lower conditions of pH, mass weight and incubation time (7 hours only). The bio-formed Au NPs were fully characterized with different techniques to study their nano-metrological measurements. The X-ray diffraction (XRD) technique and high resolution transmission electron microscopy (HR-TEM) were used to determine the crystal structure, size and shape of Au NPs. Fourier transform infrared spectroscopy (FTIR) studies were done to investigate the distinct functional groups present on the surface. The zetasizer technique was used to define the stability of biosynthesized gold nanoparticles. The antimicrobial activities of biosynthesized gold nanoparticles were studied against gram positive and gram negative bacteria at three different concentrations, 10, 30 and 50 μg/disc, using agar well diffusion method. The obtained results prove that the biosynthesized Au NPs gave different antimicrobial action which was strain dependent. All the tested bacterial strains were inhibited by the examined concentrations with variations in the inhibition zone diameters, except Candida albicans showed resistant to the biosynthesized Au NPs. The bio-formed gold nanoparticles can be used in different applications.