One of the most important roles for nanotechnology concerns is the development of optimizable experimental protocols for nanomaterials synthesis. The formation of silver nanoparticles (AgNPs) was supported by Rhizopus stolonifer MR11, which was isolated from olive oil mill soil samples. The ability of R. stolonifer MR11 to biosynthesize silver nanoparticles in response to various components of different culture media was tested. Furthermore, the conditions under which the reducing biomass filtrate was obtained, as well as the conditions of the bio-reduction reaction of AgNO3 into AgNPs, were investigated. The fungal biomass filtrate of the strain Rhizopus stolonifer MR11 was capable of converting silver nitrate into AgNPs, as evidenced by the color change of the fungal filtrates. UV-Vis spectrophotometer, TEM, Zeta potential, Zeta sizer, FT-IR, and XRD analyses were used to characterize the AgNPs. TEM analysis revealed that the silver nanoparticles were 1–35 nm in size. R. stolonifer MR11 produced the maximum AgNPs when grown for 18 hours at 36°C in media with starch and yeast extract as the sole carbon and nitrogen sources, respectively. The reducing biomass filtrate was obtained by incubating 5 g mycelial biomass in deionized water with a pH of 6 for 48 hours at 30°C. The optimal reduction conditions of the biosynthesis reaction were determined by adding 1.0 mM AgNO3 to a pH 5 buffered mycelial filtrate and incubating it for 72 hours at 33°C. The current study's findings highlighted the importance of process parameters at each stage for optimal AgNPs biosynthesis.