Healthcare associated infections (HCAIs) caused by Staphylococcus aureus are a major cause of mortality and morbidity, both locally in Australia and globally. S. aureus causes a variety of illnesses, ranging from skin and soft tissue infections through to pneumonia, endocarditis and bloodstream infections. Infection prevention and control (IPC) is critical for reducing the rates of S. aureus infection, with the use of biocidal agents such as chlorhexidine gluconate (CHG) being an important component of current IPC programs. Indeed, recent international guidelines recommend the universal use of biocides for skin decolonisation in "high-risk" hospital patients to prevent HCAIs, including those caused by S. aureus. This has led to concerns about possible "collateral damage" associated with the increasingly widespread and indiscriminate use of biocides such as CHG in our hospitals. Of particular concern is the possibility that CHG use might be associated with the emergence of antimicrobial resistance (AMR). Here we have used whole genome sequencing and Markov network analyses to determine the collateral antimicrobial resistances associated with biocide tolerance genes in S. aureus. These analyses clearly demonstrate the genetic potential for biocide-mediated co-selection of AMR in S. aureus. Furthermore, using a combination of in vitro testing and clinically relevant skin infection models, we provide compelling experimental evidence to show that the use of biocides, including CHG, can rapidly co-select for the emergence of multidrug resistant S. aureus isolates.