Phenol‐soluble modulin (PSM) is a peptide complex produced by the nosocomial pathogen Staphylococcus epidermidis that has a strong capacity to activate the human innate immune response. We developed a novel method based on liquid chromatography‐mass spectrometry (LC‐MS) to quantify the production of the individual PSM components. Each PSM peptide was abundant in most of the 76 S epidermidis strains tested. Importantly, none of the PSM components were secreted by an agr mutant strain, indicating that PSM synthesis is regulated strictly by the agr quorum‐sensing system. Furthermore, the agr mutant strain failed to elicit production of TNFα by human myeloid cells and induced significantly less neutrophil chemotaxis compared with the wild‐type strain. Thus, quorum‐sensing in S. epidermidis dramatically influenced activation of human host defence. We propose that an agr quorum‐sensing mechanism facilitates growth and survival in infected hosts by adapting production of the pro‐inflammatory PSMs to the stage of infection.