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Staphylococcus aureus (S. aureus) is a common human pathogen that causes a variety of diseases including infective endocarditis, necrotizing pneumonia and sepsis. An important aspect of S. aureus’ virulence is its ability to form biofilms, particularly following the implantation of indwelling medical devices. Biofilm infections are acutely difficult to treat due to the increased antimicrobial resistance rendered by this form of growth. Thus, there is a need to understand the molecular basis of biofilm formation to enable the development of new therapeutics. The S. aureus fibronectin-binding protein FnBPA is a cell wall anchored adhesin also able to bind fibrinogen. Fibronectin binding is mediated by 11 disordered binding repeats (FnBRs) via the tandem β-zipper mechanism and fibrinogen binding by the N2N3 subdomains of FnBPA’s A domain. Consequently, these regions of FnBPA are well characterised structurally. The A domain contains a third subdomain N1, which to date is poorly characterised and has no attributed function. The A domain of FnBPA is necessary for an FnBP-mediated mechanism of S. aureus biofilm formation. However, the molecular basis of FnBP-mediated biofilm formation is not understood. The main foci of this work are to determine the structure and function of the N1 subdomain and establish the molecular basis of FnBP-mediated biofilm formation. Nuclear magnetic resonance (NMR) spectroscopy was used to demonstrate the N1 subdomain is intrinsically disordered, but exhibits secondary structure propensity in the C-terminal region. A range of pull-down experiments identified a novel function of FnBPA’s A domain in the adherence to human endothelial cells. Adherence to host vasculature is potentially an important step in S. aureus bacteraemia and the involvement of N1 is the first evidence of functionality in this domain. A proposed mechanism of protein-mediated biofilm formation involves the chelation of Zn2+ to form intercellular protein dimers. The ability of FnBPA’s A domain to dimerise in a Zn2+-dependent manner was investigated and despite forming dimers in conditions mimicking those known to induce FnBP-mediated biofilm formation, the concentration of Zn2+ required far exceeded physiological concentrations. Therefore, interactions between the A domain and other biofilm matrix components were investigated. It was found that N1 interacts with wall teichoic acids, representing new insight into protein-mediated biofilm formation mechanisms and a novel function of the N1 subdomain.