1. Prey preference and seasonal variation in the diet of the Eurasian otter, Lutra lutra (Linnaeus, 1758), were examined by the analysis of 789 spraint samples collected along a 10-km section of a small lowland salmonid river in Serbia, with a trout farm situated in its lower part, from June 2009 to March 2012. Of particular interest was any possible impact of the otter on brown trout, which is important to investigate for preventing and managing any potential conflicts with fishers and fish farmers. 2. Fish were by far the most important otter prey, followed by crayfish. Cottus gobio Linnaeus, 1758 (the European bullhead) was the most common prey species, probably because of the combination of its abundance and easy catchability. Other prey contributed very little to the diet of the otters. Of 11 fish species present in the river, five were found in the otter diet: the European bullhead, Salmo trutta Linnaeus, 1758 (brown trout), Squalius cephalus (Linnaeus, 1758) (chub), Barbus balcanicus Kotlík, Tsigenopoulos, Ráb & Berrebi, 2002 (the Danube barbel), and Phoxinus phoxinus (Linnaeus, 1758) (the Eurasian minnow). The bullhead was the only positively selected species, however. The share of fish prey in the otter diet was stable throughout the year, except for a decrease noticed during the summer.3. The results suggest that otters on the River Gradac target smaller, sedentary, and less nutritious prey. Owing to a small percentage of brown trout in otter spraints, we surmise that otters do not represent a major biological or economic threat, either to the native trout population in the river or to the trout from the nearby fish farm.4. Although there are no reports of otters causing damage to the trout population in the local fish farm, it is advisable to establish mechanisms for the compensation of fish farmers in case such damage does occur.5. The data presented in this paper can be used to contribute to the conservation of the otter in stream habitats, especially because there are no published studies on the dietary requirements of otter in Serbia.