Abstract Artificial light at night (ALAN) is considered as a major threat to biodiversity, especially to nocturnal species, as it reduces availability, quality and functionality of habitats. However, its effects on the way species use landscape elements such as rivers are still largely understudied, especially the effect of crossing infrastructure lighting on bridges. These elements are nevertheless key commuting and foraging habitats in heavily urbanised landscapes for several taxa such as bats that are particularly affected by ALAN. We studied the effects of the illumination of facades and undersides of bridges on the relative abundance of pipistrelle bats, on their 3D distribution and their behavioural response (i.e. flight speed) close to bridges. We set-up an innovative approach based on a microphone-array to reconstruct positions and flight trajectories in 3D. We studied the effect of lighting on bats in the close proximity of six similar bridges, mostly differentiated by the presence or absence of lighting (3 lit and 3 unlit). All bridges cross the same waterway, within a uniformly and highly urbanized agglomeration (Toulouse, France). We found that bat activity was 1.7 times lower in lit sites. Bats tended to keep a larger distance, and to fly faster close to illuminated bridges. These results suggest that bridge lighting strongly reduces habitat availability and likely connectivity for bats. In that case, results call for switching off the illumination of such bridges crossing riverine ecosystems to preserve their functionality as habitats and corridors for bats.