AbstractIn semi-arid environments, the effects of irregularly distributed rainfall, flow regulation and water inter-basin transfer enhance the spread of non-native fish to the detriment of native communities. In the River Segura, since the 1980s the number of non-native fish species has progressively increased, also because of the building of water transfer facility connecting the rivers Segura and Tajo. With the aim of highlighting how man-driven changes in the diversity of fish communities affect the diet of top-predators, we compared Eurasian otterLutra lutradiet in the span of 20 years, i.e. 1997–98 vs. 2016–19. As habitat quality affects the condition of Andalusian barbelLuciobarbus sclateri, the most widespread native fish, we also compared the size of preyed barbels to point out whether human activities may have lowered their profitability to otters. Fish and introduced red swamp crayfishProcambarus clarkiiformed the bulk of otter diet in both study periods. In 2016–19 the contribution of non-native species to otter diet increased significantly, both for crayfish and fish, which included ten non-native species. Otter feeding habits faithfully mirrored the variation in the composition of the fish community and confirmed the importance of crayfish as alternative-to-fish prey in the Iberian Peninsula. The average length of preyed barbels was significantly lower in the second study period, consistently with a decline in barbel profitability for otters.