Conventional monitoring tools are seldom effective for studying the ecology of rare and elusive mammals. In the present study, we use automated ultrasound detectors to provide information about seasonal activity of the greater noctule bat (Nyctalus lasiopterus), the largest and one of least known European bats. We selected localities within Central Europe with diverse geomorphological contexts, including rivers of different sizes and mountain passes. The study demonstrates the capability of the automatic recording approach to achieve bioacoustic discrimination of this species, but also pointed to the persistent need of integrating results from automatic classification software applications with the feedback from manual approaches. The high throughput capacity of the assay proved to be efficient, and the regular occurrence of the species was identified at two localities. These locations are associated with two known and intensely used migratory corridors of winged animals going through Vltava River valley and Červenohorské Saddle in Jeseníky mountains, as illustrated also by the activity patterns of other migratory species. Together with the occurrence of spring and autumn peaks in activity, these findings are in concordance with the plesiomorphic condition in pipistrelloid bats, showing also migratory behavior, and represent further indirect evidence of migration of the greater noctule. This pattern could be facilitated by the trophic niche of the species, involving predation of migrating songbirds. Differences in phenology of migratory species observed at particular sites likely mirror position of the locality in relation to migration flyways, seasonal and geographic variation in prey availability and energy demands etc. Further application of bioacoustic monitoring and other tools is necessary to obtain detailed information about the range and movement ecology of the species in higher latitudes.