The formation of biofilms can increase pathogenic contamination of drinking water, cause biofilm-related diseases, and alter the rate of sediment erosion in rivers and coasts. Meanwhile, some biofilms have been used in moving-bed biofilm reactors (MBBRs) to degrade contaminants in wastewater. Mechanistic understanding of biofilm formation is critical to predict and control biofilm development, yet such understanding is currently incomplete. Here, we reveal the impacts of hydrodynamic conditions and surface roughness on the formation of Pseudomonas putida biofilms through a combination of microfluidic experiments, numerical simulations, and fluid mechanics theories. We demonstrate that biofilm growth is suppressed under high flow conditions and characterize the local critical velocity for P. putida biofilms to develop, which is about 50 μm/s. We further demonstrate that micron-scale surface roughness promotes biofilm formation by increasing the area of low-velocity region. Furthermore, we show that the critical shear stress, above which biofilms cease to form, for biofilms to develop on rough surfaces is 0.9 Pa, over 3 times higher than that for flat surfaces, 0.3 Pa. The results of this study will facilitate future predictions and control of biofilm development on surfaces of drinking water pipelines, blood vessels, sediments, and MBBRs.