Quorum cheating, a socio-microbiological process that is based on mutations in cell density-sensing (quorum-sensing) systems, has emerged as an important contributor to biofilm-associated infection in the leading human pathogenStaphylococcus aureus. This is because inactivation of the staphylococcal Agr quorum-sensing system leads to pronounced biofilm formation, increasing resistance to antibiotics and immune defense mechanisms. Since biofilm infections in the clinic usually progress under antibiotic treatment, we here investigated whether such treatment promotes biofilm infection via the promotion of quorum cheating. Quorum cheater development was stimulated by several antibiotics used in the treatment of staphylococcal biofilm infections more strongly in biofilm than in the planktonic mode of growth. Sub-inhibitory concentrations of levofloxacin and vancomycin were investigated for their impact on biofilm-associated (subcutaneous catheter-associated and prosthetic joint-associated infection), where in contrast to a non-biofilm-associated subcutaneous skin infection model, a significant increase of the bacterial load and development ofagrmutants was observed. Our results directly demonstrate the development of Agr dysfunctionality in animal biofilm-associated infection models and reveal that inappropriate antibiotic treatment can be counterproductive for such infections as it promotes quorum cheating and the associated development of biofilms.