The use of flow displacement systems for studying initial bacterial adhesion to surfaces is mostly confined to transparent substrata. The objective of this study was to investigate a method based on macroscopic fluorescence imaging to enumerate adhering fluorescent bacteria on non-transparent substrata, real-time and under flow. To this end, a stepwise protocol is described to quantify adhesion of green-fluorescent-protein producing Staphylococcus aureus on polished and non-polished metal and polymer surfaces accounting for surface-enhanced-fluorescence on metal surfaces, quantified by the ratio of the single cell fluorescence observed for adhering and planktonic bacteria. Enumeration of adhering fluorescent staphylococci by the proposed method is consistent with results obtained using metallurgical microscopy. An advantage however, is that the non-homogeneous surface coverage and surface roughness do not limit the applicability of the method. Moreover, the accurate quantification of surface-enhanced-fluorescence arising from adhering bacteria offers a new pathway for evaluating bacterial cell surface deformation during adhesion.