Multidrug-resistant Staphylococcus aureus (MDR S. aureus) is a critical global pathogen, causing significant morbidity and mortality in humans and animals. This pathogen’s ability to form biofilms shields it from antibiotics and facilitates the transfer of resistance genes, underscoring the urgent need for new anti-biofilm strategies. Leveraging microbial ecology and coevolution, probiotic species like Bacillus subtilis are gaining traction as potential therapies due to their ability to reduce S. aureus colonization and virulence. In this study, we isolated and screened 1123 Bacillus strains from concentrated livestock environments where frequent interactions with S. aureus were believed to occur. Subsequent screening identified B. subtilis 6D1, a strain with remarkable ability to inhibit biofilm growth, disassemble mature biofilms, and enhance the antibiotic sensitivity of S. aureus biofilms via Agr quorum sensing interference. Biochemical and molecular analyses revealed that multiple surfactin isoforms and an uncharacterized peptide were responsible for this activity. Compared to commercial HPLC-grade surfactin, B. subtilis 6D1-derived peptides more effectively inhibited biofilm formation across all four S. aureus Agr backgrounds and prevented S. aureus-induced cytotoxicity in HT29 human intestinal cells. Using adaptive laboratory evolution to understand the effects of long-term exposure to anti-biofilm compounds, we found that constant exposure to B. subtilis 6D1 peptides increased biofilm formation when exposure ceased, reduced cytotoxicity, and decreased interspecies competitiveness of S. aureus. These results suggest that while anti-biofilm compounds may reduce the likelihood of selecting for antibiotic resistance, they can still drive bacterial adaptation that promotes biofilm production and possible recalcitrance in the absence of these compounds. Our findings highlight the need to anticipate adaptive trade-offs when developing anti-biofilm therapies. Overall, this study illustrates the potential of exploiting microbial diversity to discover novel anti-biofilm agents, offering promising strategies to combat MDR S. aureus infections and enhance antibiotic efficacy in clinical and veterinary settings.