Nanomaterials derived from seaweed have developed as an alternative option for fighting infections caused by biofilm-forming microbial pathogens. This research aimed to discover potential seaweed-derived nanomaterials with antimicrobial and antibiofilm action against bacterial and fungal pathogens. Among seven algal species, the extract from Eisenia bicyclis inhibited biofilms of Klebsiella pneumoniae, Staphylococcus aureus, and Listeria monocytogenes most effectively at sub-MIC levels. As a result, in the present study, E. bicyclis was chosen as a prospective seaweed for producing E. bicyclis-gold nanoparticles (EB-AuNPs). Furthermore, the mass spectra of E. bicyclis reveal the presence of a number of potentially beneficial chemicals. The polyhedral shape of the synthesized EB-AuNP with a size value of 154.74 ± 33.46 nm was extensively described. The lowest inhibitory concentration of EB-AuNPs against bacterial pathogens (e.g., L.monocytogenes, S. aureus, Pseudomonas aeruginosa, and K. pneumoniae) and fungal pathogens (Candida albicans) ranges from 512 to >2048 μg/mL. Sub-MIC of EB-AuNPs reduces biofilm formation in P. aeruginosa, K. pneumoniae, L. monocytogenes, and S. aureus by 57.22 %, 58.60 %, 33.80 %, and 91.13 %, respectively. EB-AuNPs eliminate the mature biofilm of K. pneumoniae at > MIC, MIC, and sub-MIC concentrations. Furthermore, EB-AuNPs at the sub-MIC level suppress key virulence factors generated by P. aeruginosa, including motility, protease activity, pyoverdine, and pyocyanin, whereas it also suppresses the production of staphyloxanthin virulence factor from S. aureus. The current research reveals that seaweed extracts and a biocompatible seaweed-AuNP have substantial antibacterial, antibiofilm, and antivirulence actions against bacterial and fungal pathogens.