Given the increasing threat posed by antimicrobial-resistant microorganisms, there is a critical need for novel antimicrobial strategies. This study evaluates the antimicrobial efficacy of biogenic silver nanoparticles synthesized by Phanerochaete chrysosporium against a range of clinically and agriculturally relevant pathogens. A detailed physicochemical characterization confirmed that biogenic silver nanoparticles are small (<50 nm), highly stable, with strong negative ζ-potential. In vitro antimicrobial activity assays showed lower Minimum Inhibitory Concentrations of these nanoparticles than AgNO₃. Mechanistic studies using Confocal Raman Microscopy and Cryo-Electron Microscopy revealed that biogenic silver nanoparticles interact with microbial cell surfaces, leading to pore formation, membrane and wall disruption, and cytoplasmic disorganization. The generation of reactive oxygen species was identified as a primary antimicrobial mechanism, causing oxidative stress and damage to essential biomolecules. These effects produced by P. chrysosporium nanoparticles were observed, for the first time, in the evaluated microorganisms: Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Penicillium expansum. Finally, although nanoparticles presented a dose-dependent cytotoxicity against HT-29 and THP-1 macrophages cells, they remained non-toxic at therapeutically relevant concentrations. These findings highlight the effectiveness of biogenic nanoparticles as antimicrobial agents with multiple mechanisms of action and underscore the potential of Confocal Raman Microscopy as a powerful tool for studying biomolecular disruptions induced by antimicrobial treatments. This approach advances the molecular understanding of nanoparticle-based antimicrobials, guiding innovative strategies.