With the development of aquaculture, there is an urgent demand for an alternative antibacterial agent to reduce the drug resistance and environmental pollution caused by the abuse of antibiotics. Recently, as promising antimicrobial agents, silver nanoparticles (AgNPs) synthesized via organisms has received much attention. Cordyceps militaris, a valuable edible and medicinal fungus, is rich in a variety of biologically active substances. So, it is of interest to biosynthesis a new antimicrobial agent of AgNPs with the aid of C. militaris. In this study, AgNPs were synthesized using the cell filtrate of C. militaris mycelium as reducing agents. The formation of AgNPs was first confirmed by the surface plasmon resonance illustrated in UV-evisible spectrophotometer. X-ray diffraction analysis further confirmed that the AgNPs were composed of highly crystalline Ag. Transmission electron microscopy analysis revealed that most the AgNPs were in spherical shape with mean diameter about 15 nm. Moreover, the synthesized AgNPs exhibited superior antibacterial activities against the all the aquatic as well as clinical pathogenic bacteria. Besides, the prepared AgNPs were proven to be highly stable, even after long-term storage. To the best of our knowledge, this is the first report that the C. militaris extract could facilitate the synthesis of AgNPs. (C) 2016 Elsevier B.V. All rights reserved.