The green synthesis of metal nanoparticles has received substantial attention due to their applications in various domains. The aim of the study was to obtain silver nanoparticles (AgNPs) by green synthesis with filamentous fungi, such as Cladosporium cladosporoides, Penicillium chrysogenum, and Purpureocillium lilacinum. Fungal species were grown on nutrient media and aqueous mycelium extracts were used to reduce Ag+ to Ag (0). The silver nanoparticles were analyzed by various techniques, such as UV-Visible spectroscopy (UV-Vis), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), dynamic light scattering (DLS), and Zeta potential. The formation of silver nanoparticles was confirmed by UV-Vis spectroscopy and the color change of the mixture containing metal precursor and aqueous mycelium extract. FTIR displayed different functional groups as capping and reducing agents for the biosynthesis of AgNPs. SEM and TEM provided information on the particles’ morphology. DLS diagrams indicated mean particle diameters in the 124–168 nm region. All biosynthesized AgNPs had negative zeta values, which is a sign of good stability. Silver nanoparticles were evaluated for antimicrobial activity, and the most active were those synthesized with metabolites from Cladosporium, leading to 93.75% inhibition of Staphylococcus aureus, 67.20% of Escherichia coli, and 69.56% of Candida albicans. With the highest microbial inhibition percentage and a very good Poly Dispersion Index (Pd I), Cladosporium cladosporoides was selected as an environmentally friendly source of silver nanoparticles that could be used as a potential antimicrobial agent.