- Human pathogenic bacteria circulating in the bloodstream need to find a way to interact with endothelial cells (ECs) lining the blood vessels to infect and colonise the host. The extracellular matrix (ECM) of ECs might represent an attractive initial target for bacterial interaction, as many bacterial adhesins have reported affinities to ECM proteins, in particular to fibronectin (Fn). Here, we analysed the general role of EC-expressed Fn for bacterial adhesion. For this, we evaluated the expression levels of ECM coding genes in different ECs, revealing that Fn is the highest expressed gene and thereby, it is highly abundant in the ECM environment of ECs. The role of Fn as a mediator in bacterial cell-host adhesion was evaluated in adhesion assays of Acinetobacter baumannii, Bartonella henselae, Borrelia burgdorferi, and Staphylococcus aureus to ECs. The assays demonstrated that bacteria colocalised with Fn fibres, as observed by confocal laser scanning microscopy. Fn removal from the ECM environment (FN1 knockout ECs) diminished bacterial adherence to ECs in both static and dynamic adhesion assays to varying extents, as evaluated via absolute quantification using qPCR. Interactions between adhesins and Fn might represent the crucial step for the adhesion of human-pathogenic Gram-negative and Gram-positive bacteria targeting the ECs as a niche of infection.