Synthesis of nanoparticles by using micro-organisms is a promising future achievement in the field of green nanotechnology. In this respect, the extracellular biosynthesis of silver nanoparticles (AgNPs) by using culture supernatants of soil fungus Fusarium oxysporum was studied. The bioreduction of AgNPs was detected by ultraviolet-visible spectroscopy, and the synthesized AgNPs were characterized by transmission electron microscopy and Zeta potential. In addition, evaluation of antimicrobial activity of the synthesized AgNPs was tested. The synthesized AgNPs appeared as polydispersed spherical particles, stabilized in the solution and ranging in size from 9 to 24 nm. Also, the results showed that the synthesized AgNPs had inhibition effects on various pathogenic microorganisms including bacteria and fungi. Synthesis of AgNPs by soil fungus Fusarium oxysporum may represent a safe, non-sophisticated and economic method for production of antimicrobial agents.