Diet compositions of the European polecat (Mustela putorius) and the stone marten (Martes foina) were studied through macroscopic and microscopic analyses of 69 polecat and 120 stone marten stomachs dissected between 2000 and 2006 in Southern Moravia (Czech Republic). The diets of both mustelid species included a wide variety of prey species but were dominated by mammals and birds. Frogs were consumed only in winter. No reptiles were found in the diet of either species. Invertebrates were rarely present in the polecat diet but very common in the stone marten diet. In summer, the most common food for the stone marten was fruit. The stone marten consumed significantly more rodent species, especially rats (Rattus norvegicus), and songbirds and the food niche of the stone marten was broader than that of the polecat. The trophic niche overlap of both species, based on Pianka’s index, was highest in winter. Stone martens appear to be an important food competitor of European polecats in the Czech Republic, mainly in winter when food resources are limited.