Bacterial communities attached to surfaces under fluid flow represent a widespread lifestyle of the microbial world. Through shear stress generation and molecular transport regulation, hydrodynamics conveys effects that are very different by nature but strongly coupled. To decipher the influence of these levers on bacterial biofilms immersed in moving fluids, we quantitatively and simultaneously investigated physicochemical and biological properties of the biofilm. We designed a millifluidic setup allowing to control hydrodynamic conditions and to monitor biofilm development in real time using microscope imaging. We also conducted a transcriptomic analysis to detect a potential physiological response to hydrodynamics. We discovered that a threshold value of shear stress determined biofilm settlement, with sub-piconewton forces sufficient to prevent biofilm initiation. As a consequence, distinct hydrodynamic conditions, which set spatial distribution of shear stress, promoted distinct colonization patterns with consequences on the growth mode. However, no direct impact of mechanical forces on biofilm growth rate was observed. Consistently, no mechanosensing gene emerged from our differential transcriptomic analysis comparing distinct hydrodynamic conditions. Instead, we found that hydrodynamic molecular transport crucially impacts biofilm growth by controlling oxygen availability. Our results shed light on biofilm response to hydrodynamics and open new avenues to achieve informed design of fluidic setups for investigating, engineering or fighting adherent communities.