Nanotopography influences on bacterial adhesion were investigated with surfaces with controlled interspatial nanopillars distances. Surfaces were coated with proteins (fibrinogen, collagen, serum and saliva) prior to E. coli adhesion under flow conditions. PiFM provided chemical mapping and showed that proteins adsorbed both between and onto the nanopillars with a preference to areas between the nanopillars. E. coli adhered least to protein-coated areas with low surface nanopillar coverage, most to surfaces coated with saliva, while human serum led to the lowest adhesion. Protein-coated nanostructured surfaces affected the adhesion of E. coli.