When interacted with the aqueous environment, steel structures are usually prone to surface biofouling as the consequence of attachment and colonization of bacteria and other fouler species. Prevention of occurring of the bacterial biofouling is essential for desired performances of the structures. In most cases, formation of a conditioning layer takes place in the first place, which in turn regulates initiation and following development of biofilm. This paper presents our recent efforts on characterizing adsorbed albumin and alginate, typical protein/polysaccharide for biofouling research, and understanding the influence of the conditioning layer on formation of biofilm of typical bacteria, Escherichia coli and bacillus sp.. Conformations of adsorbed biomolecules were visualized by electron microscopy and the influence of typical divalent cations in the testing solutions on attachment and colonization of the bacteira was examined by analyzing their extracellular polymeric substances. Corrosion behaviors of stainless steel with the formation of conditioning layer/biofilm were also assessed and elucidated. In addition, effect of shear stress generated by laminar and weakly turbulent flow on adhesion of the bacteria was also investigated through n merical simulation and bacterial adhesion testing. Our results would give certain insight into constructing appropriate surface structures of stainless steel for antifouling applications.