Abstract: The method for the biosynthesis of silver sulfide nanoparticles using a strain of Shewanella oneidensis MR-1 in an aqueous solution of silver nitrate and sodium thiosulfate salts has been optimized. Optimization is associated with the biosynthesis of nanoparticles directly in the culture medium in the presence of live cells, which increases the nanoparticle yield by 15–20% and significantly shortens the entire process of obtaining nanoparticles. It was also shown that an increase in the concentration of sodium thiosulfate and silver nitrate salts from 1 mM to 10 mM increases the nanoparticle concentration in the reaction solution, whereas the nanoparticle yield in terms of the silver introduced in the reaction decreases. The nanoparticles obtained by the optimized and the conventional methods do not differ in shape, size, and chemical composition. It was shown that the efficiency of nanoparticle biosynthesis depends on the composition of the liquid nutrient medium for S. oneidensis MR-1 cell growth. Cell culturing in nutrient medium for over 24 h fails to intensify the biosynthesis process. © 2018, Pleiades Publishing, Inc.