Understanding how bacteria adhere to a surface is a critical step in the development of novel materials and coatings to prevent bacteria forming biofilms. Here, surface plasmon resonance (SPR) spectroscopy, in combination with self-assembled monolayers (SAMs) that have different backbone structures and/or functional groups, is used for the first time to study the initial stages of bacterial adhesion to surfaces (i.e., initial interaction of cells with a surface, a process governed by van der Waals, electrostatic, and hydrophobic interactions). The work highlights SPR spectroscopy as a powerful and unique approach to probe bacterial adhesion in real time. SPR spectral data reveal different kinetics of adhesion for the interaction of two marine bacterial species (Marinobacter hydrocarbonoclasticus and Cobetia marina) to a range of organosulfur SAMs. Furthermore, the extent of adhesion is dependent on the backbone structures and functional groups of the SAMs. The role of extracellular polymeric substances (EPS) in bacterial adhesion is also investigated. Pre-conditioning experiments with cell-free culture supernatants, containing planktonic EPS, allow quantification of the amount adsorbed onto surfaces and directly account for the impact of EPS adsorption on bacterial adhesion in the assay. While the physicochemical characteristics of the surfaces play a significant role in determining bacterial cell adhesion for low levels of conditioning by planktonic EPS, greater levels of conditioning by EPS reduce the difference between surfaces.