This study investigates the synthesis and properties of silver nanoparticles (AgNPs) derived from *Pseudomonas aeruginosa* pyoverdine (PVD) as a potential antibiofilm and antivirulence agent. PVD-AgNPs were synthesized using a green chemistry approach, characterized by various techniques, and evaluated for their antimicrobial activity against a range of bacterial and fungal pathogens. The MIC values of PVD-AgNPs were found to be lower in host-mimicking media (artificial sputum, saliva, and synthetic human urine) compared to standard growth media. PVD-AgNPs effectively inhibited and eradicated both initial and mature biofilms of *P. aeruginosa*, *Staphylococcus aureus*, and *Candida albicans*. They also enhanced the efficacy of antibiotics and antifungals against mature biofilms. Additionally, PVD-AgNPs attenuated several virulence traits of *P. aeruginosa*, including pyocyanin production, protease activity, hemolytic activity, and motility. Gene expression analysis revealed significant downregulation of biofilm and virulence-related genes in the presence of PVD-AgNPs. The study highlights the potential of PVD-AgNPs as a novel therapeutic agent for combating biofilm-forming microbial pathogens.This study investigates the synthesis and properties of silver nanoparticles (AgNPs) derived from *Pseudomonas aeruginosa* pyoverdine (PVD) as a potential antibiofilm and antivirulence agent. PVD-AgNPs were synthesized using a green chemistry approach, characterized by various techniques, and evaluated for their antimicrobial activity against a range of bacterial and fungal pathogens. The MIC values of PVD-AgNPs were found to be lower in host-mimicking media (artificial sputum, saliva, and synthetic human urine) compared to standard growth media. PVD-AgNPs effectively inhibited and eradicated both initial and mature biofilms of *P. aeruginosa*, *Staphylococcus aureus*, and *Candida albicans*. They also enhanced the efficacy of antibiotics and antifungals against mature biofilms. Additionally, PVD-AgNPs attenuated several virulence traits of *P. aeruginosa*, including pyocyanin production, protease activity, hemolytic activity, and motility. Gene expression analysis revealed significant downregulation of biofilm and virulence-related genes in the presence of PVD-AgNPs. The study highlights the potential of PVD-AgNPs as a novel therapeutic agent for combating biofilm-forming microbial pathogens.