Artificial light at night (ALAN) is a major form of anthropogenic pollution, disrupting nocturnal wildlife behaviour and ecosystem function. Large construction sites are typically located at the intersection of urban and natural areas, introducing intense lighting into previously dark natural habitats. This study examines the responses of bats to intense nighttime lighting at a major construction site associated with a linear transport infrastructure (LTI) project. We used passive acoustic monitoring to record bat activity and species richness at the construction site and in adjacent urban and natural areas with different lighting levels. Generalist species, such as Pipistrellus kuhlii and Nyctalus leisleri, were attracted to illuminated areas, likely due to increased prey availability. Conversely, sensitive species, such as those from the Myotis and Plecotus genera, along with Barbastella barbastellus, avoided illuminated areas, particularly the construction site. Species richness was significantly lower at the construction site compared to other environments, reflecting ALAN’s barrier effect on movements and habitat accessibility for more sensitive species. The findings highlight the ecological impacts of introducing ALAN to previously unlit natural environments, underscoring the urgent need for implementing ALAN mitigation strategies in urban planning and construction practices to protect biodiversity in urbanising areas.