Laminarin-gold nanoparticles (Lam-AuNPs) were synthesized using laminarin, a polysaccharide derived from brown algae, as a reducing agent. These nanoparticles were characterized as spherical, with a size of 49.84 ± 7.32 nm and a zeta potential of -26.49 ± 1.29 mV. Lam-AuNPs demonstrated antibiofilm and antivirulence activities against Pseudomonas aeruginosa and Staphylococcus aureus in both standard and host-mimicking media. The minimum inhibitory concentration (MIC) of Lam-AuNPs against these pathogens varied from 2 to 1024 µg/mL, with lower values observed in host-mimicking media such as artificial saliva and SHU. Sub-MIC concentrations of Lam-AuNPs significantly reduced biofilm formation and virulence factors in both media. The nanoparticles also inhibited hemolysis, pyocyanin, pyoverdine, protease, and motility in P. aeruginosa, as well as hemolysis and amyloid fibril production in S. aureus. Lam-AuNPs effectively dispersed preformed biofilms in a dose-dependent manner. The study concluded that Lam-AuNPs could serve as an effective alternative to control biofilm and virulence features of P. aeruginosa and S. aureus. The nanoparticles showed no cytotoxicity to macrophage cells, indicating their biocompatibility. The findings suggest that Lam-AuNPs have potential as antibiofilm and antivirulence agents in healthcare applications.Laminarin-gold nanoparticles (Lam-AuNPs) were synthesized using laminarin, a polysaccharide derived from brown algae, as a reducing agent. These nanoparticles were characterized as spherical, with a size of 49.84 ± 7.32 nm and a zeta potential of -26.49 ± 1.29 mV. Lam-AuNPs demonstrated antibiofilm and antivirulence activities against Pseudomonas aeruginosa and Staphylococcus aureus in both standard and host-mimicking media. The minimum inhibitory concentration (MIC) of Lam-AuNPs against these pathogens varied from 2 to 1024 µg/mL, with lower values observed in host-mimicking media such as artificial saliva and SHU. Sub-MIC concentrations of Lam-AuNPs significantly reduced biofilm formation and virulence factors in both media. The nanoparticles also inhibited hemolysis, pyocyanin, pyoverdine, protease, and motility in P. aeruginosa, as well as hemolysis and amyloid fibril production in S. aureus. Lam-AuNPs effectively dispersed preformed biofilms in a dose-dependent manner. The study concluded that Lam-AuNPs could serve as an effective alternative to control biofilm and virulence features of P. aeruginosa and S. aureus. The nanoparticles showed no cytotoxicity to macrophage cells, indicating their biocompatibility. The findings suggest that Lam-AuNPs have potential as antibiofilm and antivirulence agents in healthcare applications.