Wildlife crossing structures (WCSs) enhance connectivity between habitats of wild animals fragmented by fenced motorways, but factors affecting their use by targeted species remain understudied, particularly in areas recently recolonized by large carnivores. We investigated the use of WCS—6 overpasses (width 30-45m), 5 large underpasses (width 33–114 m) and 4 small underpasses (width 15–19 m)—located along the A4 motorway in the Lower Silesian Forest (western Poland), a large forest tract recently recolonised by wolves (Canis lupus). Identifying and counting tracks of mammals left on sand-beds as well as individuals recorded by camera traps were used to determine species diversity, number and activity patterns of mammals on WCS, and to reveal seasonal and temporal changes of WCS use over 3 years of study (2010–2013). WCSs were mostly used by wild species (51.5%), followed by humans (34.8%), livestock and pets (13.7%). Among wild species, ungulates were the most common (77.4% of crossings), while lagomorphs and carnivores were recorded less often (15% and 7.6% of crossings, respectively). The number of species and crossings of wild mammals, especially wild ungulates and wolves, was substantially higher on overpasses (mean effective number of species (Hill numbers): 0D = 7.8, 1D = 4.1 and 2D = 3.3) than on underpasses (0D = 6.3, 1D = 2.9 and 2D = 2.3) and was not affected by distance between WCS and human settlements or WCS width. There was a higher diversity of wild species and more crossings under large extended bridges than on smaller underpasses. The number of species and number of crossings of wild mammals, domestic animals and people increased from 2010 to 2013. There was a significant difference in activity patterns, with almost all wild species being nocturnal, in contrast to people and dogs. There was no relationship between crossing time and rates of wild carnivores and potential prey. We conclude that overpasses, even with steep entrance slopes (25–26.5%) or integrated with moderately used gravel roads, maintain movement of wild terrestrial mammals much better than underpasses, and the presence of wolves does not hamper the movement of other wild species. As there are significant temporal changes in use of WCS by mammals, we recommend monitoring WCS in all seasons for at least 3 years as a minimum standard for the post-investment assessment of WCS utilization by animals.