Updated project metadata. Cyanobacterial blooms associated with the benthic mat have been rising. Besides the ongoing concern about toxins production, cyanobacteria are actively involved in marine biofilms, representing several economic and environmental impacts. Proteomic studies on cyanobacterial biofilms could be an effective approach to establish metabolic pathways that affect these fouling organisms and, consequently, obtain novel control strategies against them. Currently, there are few studies in this field on filamentous cyanobacteria. Thus, standard methodologies for following cyanobacterial biofilm development for a long-term assay and a quantitative proteomics analysis were performed in this work. Biofilm development from unidentified filamentous Synechococcales LEGE 06021 was evaluated on different surfaces, glass and perspex, and at two significant shear rates for marine environments (4 s-1 and 40 s-1). Higher biofilm development was observed at 4 s-1, and these biofilms showed a lower roughness coefficient value than those formed at higher shear. Overall, about 1,877 proteins were identified, and differences in proteome were more noticeable between the two hydrodynamic conditions than those found between the two surfaces. 20 Differentially Expressed Proteins (DEPs) were found between 4 s-1 vs. 40 s-1, of which 15 DEPs were found on glass, whereas five DEPs were found on perspex. On the glass, some of these DEPs include phage tail proteins, orange carotenoid protein, enzymes like cyanophynase, glutathione-dependent formaldehyde dehydrogenase, and MoaD/ThiS family protein, while on perspex, the DEPs include enzymes such as transketolase, dihydroxy-acid dehydratase, iron ABC transporter substrate-binding protein or transcription termination/antitermination protein NusG. In summary, the biofilm structure, chlorophyll a content, total biomass, and proteomic profile are more affected by the hydrodynamic conditions than by the surfaces employed. These findings suggest that most of the metabolic changes could be produced to counterbalance the different shear rates. However, the differential expression of some proteins could be associated with the surfaces used. This study helps to consolidate the knowledge of the main factors affecting biofilm development, and sheds new lights on putative targets to address new antimicrobial strategies.