Biofouling is a specific lifestyle including both marine prokaryotic and eukaryotic communities. Hydrodynamics are poorly studied parameters affecting biofouling formation. This study aimed to investigate how water dynamics in the Etel Estuary (Northwest Atlantic coasts of France) influences the colonization of artificial substrates. Hydrodynamic conditions, mainly identified as shear stress, were characterized by measuring current velocity, turbulence intensity and energy using Acoustic Doppler Current Profiler (ADCP). One-month biofouling was analyzed by coupling metabarcoding (16S rRNA, 18S rRNA and COI genes), untargeted metabolomics (liquid chromatography coupled with high-resolution mass spectrometry, LC-HRMS) and characterization of the main biochemical components of the microbial exopolymeric matrix. A higher richness was observed for biofouling communities (prokaryotes and eukaryotes) exposed to the strongest currents. Ectopleura (Cnidaria) and its putative symbionts Endozoicomonas (Gammaproteobacteria) were dominant in the less dynamic conditions. Eukaryotes assemblages were specifically shaped by shear stress, leading to drastic changes in metabolite profiles. Under high hydrodynamic conditions, the exopolymeric matrix increased and was composed of 6 times more polysaccharides than proteins, these latter playing a crucial role in the adhesion and cohesion properties of biofilms. This original multidisciplinary approach demonstrated the importance of shear stress on both the structure of marine biofouling and the metabolic response of these complex communities.