To understand the first step of marine benthic microbial mat formation and biofouling phenomena, caused by diatoms in the marine environment, the surface properties of the epipelic diatom Navicula jeffreyi were studied and the composition of its bound Extracellular Polymeric Substances (EPS) was determined. These parameters are determining factors for the initial adhesion step of diatoms to other constituents that start marine fouling. Surface energy of a diatom cell layer was determined using the sessile drop technique and highlights that diatoms show a moderate hydrophobic character (contact angle with water >68°), no Lewis acid character (γ+ <1 mJ/m²), and a low Lewis basic character (γ− = 16.1 mJ/m²). An extraction procedure using a cationic resin subtracted only the bound EPS. Biochemical assays showed that there were 2.5 times more proteins than sugars. The propensity of Navicula jeffreyi diatom to adhere to five different solid surfaces, showing a gradient in their hydrophobic and hydrophilic character, was measured. The attachment densities were high on hydrophobic surfaces such as polytetrafluoroethylene and very low on substrata with surface free energy over 40-50 mJ/m². Using a thermodynamic approach, the free energy of adhesion of the diatom to the five substrata was determined, and led to a very strong correlation with attachment densities for polytetrafluoroethylene, polyamide, polyethylene, and stainless steel.