Adsorption of organic macromolecules onto surfaces in contact with waters forms a so-called conditioning film and induces modifications of the surface properties. Here, we characterized conditioning films formed onto two hydrophobic materials (used as pipe liner) and immersed for 24 h in tap water. Using combination of atomic force microscopy (AFM), and chemical force microscopy (CFM), we detected some changes in roughness and hydrophilic/hydrophobic balance of the surface of the tested coupons, and also the deposition of numerous organic polymers (few millions/cm2) randomly distributed on the surface. The maximum molecular extension of these organic polymers was in the range of 250–1250 nm according to the tested materials. Systematic analysis of the force curves with the theoretical models (WLC and FJC) allowed determining the proportion of rupture events related to the unfolding of both polysaccharide and polypeptide segments, which represented 75–80% and 20–25% of the analyzed curves, respectively. The number of autochthonous drinking water bacteria, which attached to the material within the same period of time was 10000-folds lower than the detected number of polymers attached to the surface. Even in drinking water systems with relatively low organic matter (dissolved organic carbon < 1.1 mg/L), the potential of formation of a conditioning biofilm is important.