Green, eco-friendly, cost-effective, safe, and reliable processes for synthesizing nanoparticles using microorganisms as bio-nanofactories have garnered significant interest due to their ability to transform metals into nanoscale particles for use in various fields. In this study, silver nitrate and supernatants from Cladosporium herbarum, isolated from water samples of Qarun Lake in Egypt, were utilized for the extracellular biosynthesis of silver nanoparticles (AgNPs), and their effectiveness against various pathogenic microorganisms was assessed. Silver nanoparticles were synthesized using four isolated marine fungi, with optimization of reaction parameters, and the resultant nanoparticles were characterized using a range of analytical techniques including Transmission Electron Microscopy (TEM), Fourier transform infrared (FT-IR), and X-ray analysis. The antimicrobial activity of the biosynthesized silver nanoparticles was evaluated against a variety of pathogens, including both Gram-positive and Gram-negative bacteria, yeast, and fungi. The results demonstrated that silver nanoparticles synthesized by Cladosporium herbarum exhibited potent antimicrobial activity against different pathogens under optimal conditions, including a substrate concentration of 5 mM, an incubation temperature of 37°C, a pH value of 6, and a 24-hour incubation period. TEM analysis revealed the formation of spherical, well-dispersed nanoparticles with sizes ranging from 4 to 23.1nm, while FT-IR spectroscopy indicated prominent absorption bands at 556, 762, 1349, 1451, 1562, 1620, and 3352 cm⁻¹, corresponding to the stretching vibrations of alkyl halides, C-H groups, phenolic O-H groups, amide C-N groups, NH₂ groups, N-H groups, and hydroxyl (OH) groups, respectively. The biosynthesis of silver nanoparticles mediated by Cladosporium herbarum holds promise as a potential antimicrobial agent against various pathogenic microbes.