The biosynthesis of silver nanoparticles (AgNPs) by microorganisms has become a hot topic in recent years, although its mechanism is still not well understood. Here we report the extracellular biosynthesis of AgNPs by the fungus Fusarium oxysporum through a superoxide-dependent mechanism. Reduction of Ag + to AgNPs in the extracellular region of F. oxysporum was verified by transmission electron microscopy, while the superoxide produced extracellularly by F. oxysporum was evidenced by chemiluminescence. We further demonstrated that the biosynthesis of AgNPs was inhibited by a superoxide scavenger or the inhibitor of NADH oxidases, and the addition of NADH significantly improved the formation of AgNPs. These results demonstrated that, for the first time, the fungus F. oxysporum can mediate the synthesis of AgNPs through the enzymatic generation of extracellular superoxide, which is helpful in understanding the biosynthesis of AgNPs and the biomineralization and transformation of silver and other metals or metalloids.