Biogenic synthesis based on microorganisms and plants utilizes biomolecules and metabolites that act as reducing and stabilizing agents. Furthermore, these biomolecules confer enhanced properties, increasing their effectiveness as antibacterial agents, even against multidrug-resistant bacteria. However, there is currently a lack of knowledge regarding the characteristics of the biomolecules involved and their mechanisms during synthesis, which limits their wider application. This research study focused on the synthesis of AgNPs using extracellularly secreted biomolecules from the filamentous fungus R. stolonifer. The results indicated that the concentration of the biomolecules depends on the response of the fungus to specific operational conditions. Furthermore, a relationship was found between the concentrations of total proteins and polyphenols, an optimal protein-to-polyphenol ratio, and the formation of AgNPs. Characterization of the fungus extracts revealed a complex mixture of over 100 metabolites, primarily phenolic compounds, proteins, and methoxylated compounds, thereby identifying a synergistic mechanism that enables the reduction and stabilization of AgNPs. Disinfection tests confirmed the antibacterial efficacy of all biosynthesized AgNPs, achieving 100% elimination of Escherichia coli and Staphylococcus aureus. The study outcomes highlight the important role of fungal biomolecules for creating a sustainable and efficient method for synthesizing antimicrobial nanoparticles with high efficacy for disinfectant applications.