Staphylococcus aureus is a versatile pathogen which can cause biofilm-associated infections with a high morbidity and mortality. S. aureus in a biofilm has a reduced metabolic activity which reduces the effectivity of antibiotics, even when the bacteria are found to be susceptible to these antibiotics in a planktonic phenotype, such as beta lactam antibiotics. Furthermore, bacteria within a biofilm are enclosed by an extracellular matrix which protects them from bacterial threats such as host antimicrobial peptides or antibiotics. There is a knowledge gap conserning the interaction of S. aureus and the host particularly during the early stages of biofilm formation. Therefore, in this thesis, we studied the interactions during these stages of biofilm formation by detemining how S. aureus cells interact with the innate immune system of the host during biofilm formation, in particularly interaction with polymorphonuclear cells under conditions that resemble the in vivo situation as close as possible. Furthermore, we developed a reliable way to detect biofilm tolerance towards antibiotics using isothermal microcalorimetry. This diagnostic method is an addition to susceptibility testing of biofilm-associated bacteria, which is currently suboptimal in most clinical microbiological laboratories. The studies in this thesis are a step towards determining which antibiotic drug or combinations of drugs are valuable in treating biofilm-associated S. aureus infections.