The recovery and valorization of metals and rare earth metals from wastewater are of great importance to prevent environmental pollution and recover valuable resources. Certain bacterial and fungal species are capable of removing metal ions from the environment by facilitating their reduction and precipitation. Even though the phenomenon is well documented, little is known about the mechanism. Therefore, we systematically investigated the influence of nitrogen sources, cultivation time, biomass, and protein concentration on silver reduction capacities of cell-free cultivation media (spent media) of Aspergillus niger, A. terreus, and A. oryzae. The spent medium of A. niger showed the highest silver reduction capacities with up to 15 μmol per milliliter spent medium when ammonium was used as the sole N-source. Silver ion reduction in the spent medium was not driven by enzymes and did not correlate with biomass concentration. Nearly full reduction capacity was reached after 2 days of incubation, long before the cessation of growth and onset of the stationary phase. The size of silver nanoparticles formed in the spent medium of A. niger was influenced by the nitrogen source, with silver nanoparticles formed in nitrate or ammonium-containing medium having an average diameter of 32 and 6 nm, respectively.