Silver nanoparticles (AgNPs) synthesized using pyoverdine (PVD) from Pseudomonas aeruginosa exhibit antibiofilm and antivirulence properties. PVD, a siderophore produced by P. aeruginosa, was used as a biological corona to synthesize AgNPs, which target microbial pathogens due to recognition by the pathogenic membrane. The synthesized PVD-AgNPs had an average size of 251.87 ± 21.8 nm and a zeta potential of -36.51 ± 0.69 mV. The minimum inhibitory concentration (MIC) of PVD-AgNPs against various pathogens was significantly lower in host-mimicking media compared to standard growth media. PVD-AgNPs effectively inhibited both initial and established biofilms of microbial pathogens, including P. aeruginosa, Listeria monocytogenes, Staphylococcus aureus, Streptococcus mutans, Escherichia coli, and Candida albicans. They also reduced virulence factors such as protease activity, motility, and production of PVD and pyocyanin in P. aeruginosa. PVD-AgNPs enhanced the susceptibility of tetracycline, PVD, and amphotericin B towards biofilms of S. aureus and C. albicans. Additionally, PVD-AgNPs downregulated the expression of biofilm and virulence-related genes in P. aeruginosa. The study highlights the potential of PVD-AgNPs as an effective alternative to traditional antibiotics for combating biofilm-associated infections.Silver nanoparticles (AgNPs) synthesized using pyoverdine (PVD) from Pseudomonas aeruginosa exhibit antibiofilm and antivirulence properties. PVD, a siderophore produced by P. aeruginosa, was used as a biological corona to synthesize AgNPs, which target microbial pathogens due to recognition by the pathogenic membrane. The synthesized PVD-AgNPs had an average size of 251.87 ± 21.8 nm and a zeta potential of -36.51 ± 0.69 mV. The minimum inhibitory concentration (MIC) of PVD-AgNPs against various pathogens was significantly lower in host-mimicking media compared to standard growth media. PVD-AgNPs effectively inhibited both initial and established biofilms of microbial pathogens, including P. aeruginosa, Listeria monocytogenes, Staphylococcus aureus, Streptococcus mutans, Escherichia coli, and Candida albicans. They also reduced virulence factors such as protease activity, motility, and production of PVD and pyocyanin in P. aeruginosa. PVD-AgNPs enhanced the susceptibility of tetracycline, PVD, and amphotericin B towards biofilms of S. aureus and C. albicans. Additionally, PVD-AgNPs downregulated the expression of biofilm and virulence-related genes in P. aeruginosa. The study highlights the potential of PVD-AgNPs as an effective alternative to traditional antibiotics for combating biofilm-associated infections.