An efficient and eco-friendly process for the synthesis of Au-Ag alloy nanoparticles of varying composition by the fungus Fusarium oxysporum was demonstrated. When fungal strains of F. oxysporum of different biomass concentrations are exposed to equimolar solutions of HAuCl4 and AgNO3, the formation of highly stable Au-Ag alloy nanoparticles of varying mole fractions were achieved. A chemical analysis of a drop-coated film of a nano Au-Ag alloy solution exposed to fungus biomass for 96 h on Si-(111) was carried out by XPS. The transmission electron microscopy (TEM) was used to analyze the size and electron density of the alloy nanoparticles obtained from the drop-coated films. It was observed that the amount of cofactor NADH plays an important role in determining the nanoalloy composition. The results show that alloyparticles, which are synthesized extracellularly, are quite stable in solution.