In this study, at first, the biosynthesis of silver nanoparticles (Ag NPs) using an aqueous extract of ThymbraspicataL wasstudied andthen, the shape and size ofthe synthesized NPs were characterized by different analysis such as UV-Vis, TEM, SEM, EDX, XRD, DLS and FT-IR. The synthesized Ag NPs were evaluated for their antibacterial activity against Escherichia coli (PTTC 1707), Staphylococcus aureus (PTTC 1112), Bacillus cereus (PTTC 1154) and Aspergillus Niger (PTTC 5012) and Candida albicans (PTTC5027) by a well-diffusion method and determination of minimum inhibitory concentration (MIC). Ag NPs displayed significant antimicrobial activity against the specimens, and they prevented the growth of bacteria and fungi at low concentrations.