This investigation aimed to characterise conditioning layers formed on AISI 316 stainless steel by different types of extracellular polymeric substances (EPS), i.e. biofilm, planktonic and capsular exopolymers, isolated from continuous cultures of marine Pseudomonas received from the National Collection of Industrial and Marine Bacteria (strain NCIMB 2021). Colorimetric assays and gas chromatography‐mass spectrometry analysis confirmed previously obtained results based on a FTIR and SDS‐PAGE study of Pseudomonas NCIMB 2021 EPS demonstrating the presence of protein, neutral and amino sugars and uronic acids. The content and the ratio of these macromolecules differed depending on the type of EPS. X‐ray photoelectron spectroscopy revealed that conditioning layers formed upon exposure of steel to EPS solutions were chemically dissimilar. It is proposed that the observed difference in the chemistry of conditioning layers is the likely reason for reported differences in attachment of Pseudomonas cells to EPS‐conditioned steel surfaces.