This study aimed to assess the actual use of overpass-type migration structures by various groups of fauna (large and medium-sized mammals, small mammals, reptiles, bats, and butterflies) and to verify the suitability of individual monitoring methods, such as camera traps, live-capture traps, monitoring squares, and detectors. Functionality is defined as the ability of the structure to enable the target species to cross the barrier safely. From a set of 36 overpasses located in the Czech Republic, objects suitable for monitoring were selected for each animal group during a screening field visit. The results showed that large and mediumsized mammals, dominated by European roe deer (Capreolus capreolus) and European fallow deer (Dama dama) actively use ecoducts, with the frequency of crossings significantly influenced by the location of the structure in the landscape, the attractiveness of the surrounding habitats, and the availability of food, rather than solely by technical parameters. At the same time, anthropogenic disturbance, which affects the daily rhythm of fauna migration, was also recorded. The monitoring data serves as the basis for the methodology being prepared to evaluate the effectiveness of migration passages and for a new database of migration objects of the Nature Conservation Agency of the Czech Republic.