In this study, an acidophilic actinobacteria strain was used as a novel reducing agent for a single-step synthesis of nanostructure silver particles. We used a Streptacidiphilus durhamensis HGG16n isolate for efficient synthesis of bioactive silver nanoparticles [bio(AgNPs)] in an inexpensive, eco-friendly, and nontoxic manner. The obtained bio(AgNPs) exhibited unique physicochemical and biochemical properties. Structural, morphological, and optical properties of the synthesized biocolloids were characterized by spectroscopy, dynamic light scattering, and electron microscopy approaches. The antimicrobial activity was evaluated using the well- and disc-diffusion methods. The obtained crystalline structure and stable biosynthesized silver nanoparticles ranged in size from 8 nm to 48 nm and were mostly spherical in shape. Antimicrobial assays of the silver nanoparticles against pathogenic bacteria showed the highest antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus, and Proteus mirabilis, followed by Escherichia coli, Klebsiella pneumoniae, and Bacillus subtilis. Moreover, the synergistic effect of bio(AgNPs) with various commercially available antibiotics was also evaluated. These results provide insight into the development of new antimicrobial agents along with synergistic enhancement of the antibacterial mechanism against clinical bacteria.