This study investigates the antibiofilm and antivirulence activities of laminarin-gold nanoparticles (Lam-AuNPs) against *Pseudomonas aeruginosa* and *Staphylococcus aureus*. Lam-AuNPs, synthesized from marine-derived laminarin, were characterized using various techniques, including FTIR, XRD, and SEM. The MIC values of Lam-AuNPs against drug-resistant microbial pathogens were determined in standard and host-mimicking media, showing lower MIC values in artificial saliva and synthetic human urine (SHU) compared to tryptic soy broth (TSB). Lam-AuNPs effectively inhibited biofilm formation in both media, with maximum inhibition at a concentration of 128 μg/mL. The sub-MIC levels of Lam-AuNPs reduced hemolysis, pyocyanin production, protease activity, and motility in *P. aeruginosa*. Additionally, Lam-AuNPs inhibited amyloid fibril formation and hemolysis in *S. aureus*. The biocompatibility of Lam-AuNPs was confirmed by a cell cytotoxicity assay, showing no cytotoxic effects at concentrations used for antibiofilm and antivirulence activities. These findings suggest that Lam-AuNPs could be a promising alternative for controlling *P. aeruginosa* and *S. aureus* infections.This study investigates the antibiofilm and antivirulence activities of laminarin-gold nanoparticles (Lam-AuNPs) against *Pseudomonas aeruginosa* and *Staphylococcus aureus*. Lam-AuNPs, synthesized from marine-derived laminarin, were characterized using various techniques, including FTIR, XRD, and SEM. The MIC values of Lam-AuNPs against drug-resistant microbial pathogens were determined in standard and host-mimicking media, showing lower MIC values in artificial saliva and synthetic human urine (SHU) compared to tryptic soy broth (TSB). Lam-AuNPs effectively inhibited biofilm formation in both media, with maximum inhibition at a concentration of 128 μg/mL. The sub-MIC levels of Lam-AuNPs reduced hemolysis, pyocyanin production, protease activity, and motility in *P. aeruginosa*. Additionally, Lam-AuNPs inhibited amyloid fibril formation and hemolysis in *S. aureus*. The biocompatibility of Lam-AuNPs was confirmed by a cell cytotoxicity assay, showing no cytotoxic effects at concentrations used for antibiofilm and antivirulence activities. These findings suggest that Lam-AuNPs could be a promising alternative for controlling *P. aeruginosa* and *S. aureus* infections.