This study modified and integrated a bioimaging method of hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) into a pipeline for assessing biofouling on stainless steel (SS). A modified protocol of double-labeling HCR-FISH was directly applied to two surface types of SS grade EN 1.4404 to detect localized bacteria and sulfate-reducing bacteria (SRB) by targeting bacterial 16 S rRNA genes and dissimilatory sulfite reductase (dsrB) genes, respectively. The protocol was first validated using microbial pure cultures and materials before being integrated into a biofouling assessment pipeline of SS in a laboratory-scale brackish water circuit, incorporating electrochemical, surface, and molecular biology characterization analyses. The double-labeling HCR-FISH improved bioimaging of surface biofilm morphology and microbial distribution, surpassing monochrome staining methods. This method was compatible and complemented other microscopy techniques and molecular biological analyses, providing additional insights into the biofilms and deposits on the alloy surfaces. The implemented assessment pipeline for biofouling determination frequently detected the ennoblement phenomenon in the evolution of marine biofilm on SS surfaces. However, within the experimental timeframe, microbial activities in the brackish seawater circuit did not flourish significantly, resulting in minimal impact on the steel material. Additionally, surface type and roughness may correlate with microbial adhesion, biofilm growth, and the deformation of passivation layers in SS. Despite abundant sessile bacteria, particularly opportunistic microorganisms, on the steel surfaces, no direct correlations with biodeterioration phenomena or influences of surface roughness of an alloy and the presence of biofilm were conclusively established.