This study describes changes in the prevalence of Leptospira interrogans infections among small mammals, including rats and larger domestic and wild mammals in Lviv Oblast, a region in western Ukraine from 2001–2015, using the microscopic agglutination test (MAT). A total of 439,948 domestic or wild animals were tested. We found the prevalence of Leptospira interrogans exposure varied among tested species and changed over the time. Infection was significantly less common in domestic animals, than in wild rodents. In swine the overall seroprevalence was 0.51%, while in cattle it was 0.19%. In dogs it was higher—2.75%. After 2006, evidence of infection was only observed in swine among domestic animals. The prevalence among large wild animals (0.25%) was similar to that among domestic animals. Among small mammals and rats, seroprevalence was most commonly observed among Rattus norvegicus (18.44%) and it was less common among other wild small mammals (8.74%). There were two dominant serogroups among large wild and domestic animals–L. icterohaemorrhagiae and L. hebdomadis while among wild small mammals the two most common were L. icterohaemorrhagiae and L. grippotyphosa. Wild animals with antibodies were found throughout the entire oblast.