The influence of surface chemistry—substrates with controlled surface density of −OH groups—on fibronectin (FN) conformation and distribution is directly observed by atomic force microscopy (AFM). FN fibrillogenesis, which is known to be a process triggered by interaction with integrins, is shown in our case to be induced by the substrate (in absence of cells), which is able to enhance FN–FN interactions leading to the formation of a protein network on the material surface. This phenomenon depends both on surface chemistry and protein concentration. The level of the FN fibrillogenesis was quantified by calculating the fractal dimension of the adsorbed protein from image analysis of the AFM results. The total amount of adsorbed FN is obtained by making use of a methodology that employs Western blotting combined with image analysis of the corresponding protein bands, with the lowest sensitivity threshold equal to 15 ng of adsorbed protein. Further, FN adsorption is correlated to human osteoblast adhesion through morphology and actin cytoskeleton formation. Actin polymerization is in need of the formation of the protein network on the substrate's surface. Cell morphology is more rounded (as quantified by calculating the circularity of the cells by image analysis) when the degree of FN fibrillogenesis on the substrate is lower.