Biofilms in drinking water distribution systems (DWDS) could exacerbate the persistence and associated risks of pathogenic Legionella pneumophila (L. pneumophila), thus raising human health concerns. However, mechanisms controlling adhesion and subsequent detachment of L. pneumophila associated with biofilms remain unclear. We determined the connection between L. pneumophila adhesion and subsequent detachment with biofilm physical structure characterization using optical coherence tomography (OCT) imaging technique. Analysis of the OCT images of multispecies biofilms grown under low nutrient condition up to 34 weeks revealed the lack of biofilm deformation even when these biofilms were exposed to flow velocity of 0.7 m/s, typical flow for DWDS. L. pneumophila adhesion on these biofilm under low flow velocity (0.007 m/s) positively correlated with biofilm roughness due to enlarged biofilm surface area and local flow conditions created by roughness asperities. The preadhered L. pneumophila on selected rough and smooth biofilms were found to detach when these biofilms were subjected to higher flow velocity. At the flow velocity of 0.1 and 0.3 m/s, the ratio of detached cell from the smooth biofilm surface was from 1.3 to 1.4 times higher than that from the rough biofilm surface, presumably because of the low shear stress zones near roughness asperities. This study determined that physical structure and local hydrodynamics control L. pneumophila adhesion to and detachment from simulated drinking water biofilm, thus it is the first step toward reducing the risk of L. pneumophila exposure and subsequent infections.