In Quebec, Canada, 5 out of the 8 species of bats are considered potentially threatened or vulnerable. Measuring the impact on bat species of environmental changes brought about by urban development is crucial for bat species conservation. At which spatial scale does a gradient of increasing urbanization have the most significant effect on the distribution and activity of bats? To address this question, 3 sampling points in each of 24 green spaces (of different sizes, degree of “naturalness”, and presence or not of water) across Montréal Island were sampled over 3 separate nights in June and July 2006. Echolocation calls of bats were recorded with Anabat detectors. Bat activity was determined by species or group of species for each green space. Various habitat factors associated with the urban gradient were acquired using GIS along a range of spatial scales, from local to landscape scales (areas of 0.1 km to 2 km radius around sampling points). Results show that patterns of distribution and bat activity differ according to species. Bats of the Myotis genus and Perimyotis subflavus were found mostly in areas with a high percentage of forest cover and near running water. Eptesicus fuscus and Lasiurus cinereus appeared to be less selective, as they were distributed much more uniformly across the study area. The more local spatial scales (from 0.1 km to 0.5 km radius) seem to have had a predominant influence on habitat requirements of bats, particularly for forest-dwelling species.