Biofilm-associated infections are a significant cause of morbidity and death. Staphylococci, above all Staphylococcus aureus and S. epidermidis, are the most frequent causes of biofilm-associated infections on indwelling medical devices. Although the mechanistic basis for the agglomeration of staphylococcal cells in biofilms has been investigated in great detail, we lack understanding of the forces and molecular determinants behind the structuring of biofilms and the detachment of cellular clusters from biofilms. These processes are of key importance for the formation of vital biofilms in vivo with the capacity of bacterial dissemination to secondary sites of infection. Recent studies showed that the phenol-soluble modulins, surfactant peptides secreted by staphylococci in a quorum-sensing controlled fashion, structure biofilms in vitro and in vivo and lead to biofilm detachment with the in vivo consequence of bacterial dissemination. These findings substantiate that quorum sensing and surfactants have widespread importance for biofilm maturation processes in bacteria and establish a novel theory of the molecular determinants driving dissemination of biofilm-associated infection.