Biofilm formation is the crucial virulence mechanism of the nosocomial pathogen Staphylococcus epidermidis. Biofilm is assembled in a multistep process starting with bacterial attachment to artificial surfaces (like polystyrol) or host tissues (like endocardium) followed by production of a matrix that mediates interbacterial adhesion and -organisation. Although polysaccharide intercellular adhesin (PIA) has long been considered an essential component of S. epidermidis biofilm there is growing evidence for PIA-independent mechanisms. We found that icaADBC-negative S. epidermidis 1585-Ra forms a PIA-independent biofilm which, as detected by CLSM, markedly differs from PIA-dependent biofilms produced by S. epidermidis 1457. By transposon mutagenesis biofilm-negative mutants were selected that all carried transposon insertions within the gene encoding for extracellular matrix binding protein (Embp). Embp is a giant (calculated MW 1 MDa) cell surface protein which consists of 59 FIVAR- and 38 GA-domains and binds to immobilized fibronectin. Embp proved both necessary and sufficient for biofilm formation in S. epidermidis 1585-Ra on polystyrol because specific anti-Embp antibodies blocked biofilm formation and a recombinant fragment of Embp composed of FIVAR- and GA-domains (rEmbp6559) induced biofilm formation in Embp-negative S. epidermidis strains. Furthermore, binding assays showed that Embp binds to immobilized fibrinogen and fibronectin via its FIVAR domains. Interestingly, in contrast to fibronectin binding protein A of S. aureus, Embp does not mediate binding to soluble plasma fibronectin. As a consequence, Embp expressing bacteria were not capable of invading primary endothelial cells, but readily adhered to extraendothelial matrix. Thus, Embp of S. epidermidis is a multifunctional cell surface factor that mediates both attachment to host extraendothelial matrix and biofilm accumulation, and therefore is well suited for promoting vascular infections like endocarditis.