In the catchment of the River Segura (SE Spain), ongoing Eurasian otter (Lutra lutra) recovery and concurrent spread of human settlements are resulting in increasing otter records into urban areas. We analysed otter diet along a natural-to-urban gradient, with the main aim of assessing to which extent man-driven variation in food availability affects its feeding habits and how this secretive mustelid can thrive in apparently unsuitable habitats. From 2016 to 2019, we collected 521 spraints, monitoring 37 sampling stations (mean length ± SD = 0.5 ± 0.3 km). In natural areas, the percent cover of undisturbed banks was the highest, with the lowest number of water abstraction systems, whilst water pollution was the highest in urban areas. Introduced red swamp crayfish (Procambarus clarkii) and fish formed the bulk of otter diet. The two most preyed fish, Luciobarbus sclateri and Pseudochondrostoma polylepis, were among the most abundant species in the river, confirming the generalist feeding behaviour of the otter. The contribution of nonnative fish was on average low, while invasive crayfish exceeded the overall importance of fish in all habitats except urban areas. Overall, diet data did not allow attesting food as a limiting resource for otters in urban areas. In contrast, marking intensity varied consistently with the natural-to-urban gradient (from 0.31 to 0.65 spraints/100 m in urban-and natural areas, respectively), suggesting that human disturbance (i.e. habitat modification and water pollution) may play a major role in shaping otter distribution.