Fungal biogenic production of silver nanoparticles (AgNPs) is a promising trend due to their facile handling and high metal tolerance. Moreover, they release a lot of extracellular molecules, which help to keep the nanoparticles in a steady form. Fusarium oxysporum (F. oxysporum) has a prolonged life span in the soil. It is one of the main causes of agricultural plants wilting in several commercially significant crops. In this investigation, a fungus was isolated from Minyat Al Nasr in Egypt, identified by traditional and molecular procedures as Penicillium expansum (P. expansum), and deposited in the gene bank with a code of PQ084992. P. expansum produced silver nanoparticles, which could be seen by a change in color. It has a notable peak at 420 nm through testing by UV spectrophotometry. Nanoparticles have been examined by electron microscopy, revealing their size range as 40-80 nm. The characterization has been completed using XRD, EDX, and FTTR. The myco-synthesized silver nanoparticles had anti-F. oxysporum with an inhibition zone of 23.1±1.2 mm, and its MIC was 15.6±0.6 μg/mL. This anti-F. oxysporum is produced by altering the ultrastructure of F. oxysporum when using the produced wilt disease in plants. Nanoparticles in relation to amphotericin B. Silver nanoparticles were tested on Vero cells and showed minimal cytotoxicity with IC50 = 165.0±2.6 μg/mL. These results suggest the potential of applying myco-synthesized silver nanoparticles against fungal pathogens.