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T HERE is an increasing worldwide demand for seafood due to the awareness of fish as an important protein source for a growing population. However, the diseases caused by multi-drug resistant (MDR) bacterial infections and the low survival rate of the fish represent significant challenges to fish farmers. The virulence and pathogenicity of bacteria are often enhanced when growing as a biofilm. Therefore, a variety of new antimicrobial drugs has attracted wide attention in treating fish pathogen bacteria. Herein, the efficiency of macroalgal extracts as an antibacterial agent against MDR Staphylococcus aureus bacteria isolated from Nile tilapia (Oreochromis niloticus) was studied. Out of 200 bacterial isolates, 50 strains were identified as S. aureus. Of these strains, 37 were MDR and showed a potential role in the production of virulence factors, including staphylokinase (70.2%), lecithinase (81%), protease (56.7%) and lipase (59.4%). Significant production of biofilm virulence factor by MDR S. aureus strains was also observed from the quantitative and qualitative analysis. Four algal species namely Jania rubens, Ulva lactuca, Sargassum vulgare, and Sargassum fusiforme were tested for their antibacterial activity against MDR S. aureus strains. Of those, S. vulgare diethyl ether extract showed the highest antibacterial. In addition, GC-MS analysis revealed 20 identified components in S. vulgare diethyl ether extract, in which Longifolene was dominant (16.5%). This study thus established the possibility of developing an antibacterial agent to combat developing MDR S. aureus and biofilm-related infections in Nile tilapia.