The biosynthesis of silver nanoparticles (Ag NPs) could play a significant role in the development of commercial antimicrobials. Herein, the biosynthesis of Ag NPs was studied using the edible mushroom Pleurotus floridanus, and following its formation, spectrophotometry was used to detect the best mushroom content, pH, temperature, and silver concentration. After that, the morphology was described via transmission electron microscopy (TEM), and nanoscale-size particles were found ranging from 11 to 13 nm. The best conditions of Ag content and pH were found at 1.0 mM and 11.0, respectively. In addition, the best mushroom extract concentration was found at 30 g/L. According to XRD analysis, the crystal structure of the formed amorphous Ag NPs is cubic with a space group of fm-3m and a space group number of 225. After that, the function groups at the surface of the prepared Ag NPs were studied via FTIR analysis, which indicated the presence of C=O, C-H, and O-H groups. These groups could indicate the presence of mushroom traces in the Ag NPs, which was confirmed via the amorphous characteristics of Ag NPs from the XRD analysis. The prepared Ag NPs have a high impact against different microorganisms, which could be attributed to the ability of Ag NPs to penetrate the cell bacterial wall.