The present study focused on the evaluation of a nonspecific synergistic effect of biogenic silver nanoparticles (AgNPs) in combination with biosurfactants against environmental bacteria and fungi. The AgNPs were synthesized in the culture supernatants of the biosurfactant producer Bacillus subtilis grown in brewery effluent, molasses or Luria-Bertani media. Antibacterial activities were tested against Gram-positive and Gram-negative bacteria, while the antifungal activity was tested against phytopathogens. The interactions between biogenic AgNPs and DNA were investigated using a cryo-TEM technique. The presence of biosurfactant significantly increased the stability of biogenic AgNPs and enhanced their antimicrobial activities. The physical properties and antimicrobial activity of biogenic AgNPs were compared with chemically synthesized Ag nanoparticles. Biogenic silver nanoparticles showed a broad spectrum of activity against bacteria and fungi. They were most active against phytopathogenic fungi and Gram-positive bacteria and less active against Gram-negative bacteria. The nonspecific synergistic effect of biogenic AgNPs and biosurfactant on the phytopathogenic fungi was especially observed. In this report, the new roles of biosurfactants as a biogenic AgNPs stabilizer and enhancer of their antimicrobial properties are presented. Our results revealed that the biologically synthesized AgNPs by the biosurfactant-producing bacterium Bacillus subtilis grown on agro-industrial wastes, such as molasses and brewery effluent, could be used as a promising new nanoagent against microbes.