Owl diets undergo qualitative changes across the different regions of their area of distribution. During the four winters (from 2014–15 to 2017–18), Long-eared Owls’ pellets were collected at three winterroosts located at the southern part of Pannonian Plain, in the Serbian province of Vojvodina. In 8070 prey items from pellets, we identified 16 mammal and 32 bird species. The Common Vole was the dominant prey species with a proportion in a range from 27.4% to 71.6%. The Muridae family formed a supplementary part of the diet: Mus sp., wood mouse and harvest mouse, during all winters. Birds were also a major supplementary prey during winter 2014–15, comprising 10.6%. A comparison of our results with the diet of Long-eared Owls wintering at the northern Pannonian plain (southwestern Slovakia) indicated an increase the proportion of some species in the southern part. How different land uses in agriculture and environmental conditions may be reflected in the food supply are discussed in relation to the diet composition of Long-eared Owls and an environment whose is richer provides both growing diversity in the diet of these owls and an expansion of their food niche. Our study described the Long-eared Owl as opportunistic predators expanding their food niche in the presence of diversified prey.