Staphylococcus aureus is the leading cause of fatal bloodstream infections, in large part due to its ability to disseminate around the body, causing complications such as endocarditis, osteomyelitis and deep tissue abscesses. These infections frequently have a biofilm component and are extremely challenging to treat as they protect the bacteria within from antibiotics and the immune system. The staphylococcal cell envelope is crucial for biofilm formation and exposure to human serum induces major structural changes in the cell wall. However, how these changes affect biofilm formation is unknown. Here, we show that incubation of S. aureus in human serum triggers increased biofilm formation in laboratory strains and in clinical isolates. This increased biofilm formation is due to specific activation of the GraRS and SaeRS two component signalling systems by serum, as well as the non-specific binding of human serum proteins to the staphylococcal surface. Activation of these stress responses results in changes in the cell wall including increased wall teichoic acids and altered surface charge, enhancing adhesion and biofilm formation. Additionally, exposure to serum significantly reduces the daptomycin susceptibility of the biofilms, compared to biofilms formed in the absence of serum, possibly contributing to the high rates of treatment failure observed with this last resort antibiotic.  Finally, serum-induced biofilm formation can be inhibited with existing antibiotics, including the cell wall synthesis inhibitor fosfomycin, providing a viable therapeutic approach to prevent biofilm formation and improve treatment outcomes in invasive staphylococcal infections.