Probiotic nutrition is frequently claimed to improve human health. In particular, live probiotic bacteria obtained with food are believed to reduce pathogen colonization and thus, susceptibility to infection. However, the underlying mechanisms remain poorly understood. Here, we report that the consumption of probiotic Bacillus bacteria comprehensively abolishes colonization with the dangerous pathogen, Staphylococcus aureus. We discovered that the widespread fengycin class of Bacillus lipopeptides achieves colonization resistance by inhibiting the S. aureus Agr quorum-sensing signaling system. Our study presents a detailed molecular mechanism underlining the importance of probiotic nutrition in reducing infectious disease. Notably, we provide human evidence supporting the biological significance of probiotic bacterial interference and show for the first time that such interference can be achieved by blocking a pathogen’s signaling system. Furthermore, our findings suggest a probiotic-based method for S. aureus decolonization and new ways to fight S. aureus infections.