Feeding ecology of a species is the result of its evolutionary history, biology, physiology and local constraints, such as prey availability, intra- and inter-specific interactions and environmental characteristics. In this study we investigated the still unknown diet of the Sicilian pond turtle, with special emphasis to the relationships with recently introduced alien species in the “Lake Preola ... [Show full abstract] and Gorghi Tondi” Nature Reserve (Sicily, Italy). A total of 83 faecal samples were collected in three different periods. Emys trinacris seems an opportunistic and generalist species. The main prey taxa were aquatic invertebrates, including the invasive Procambarus clarkii, while non-aquatic preys are found sporadically. Plant matter, mainly leaves and roots of aquatic forms, was also found in high frequency with a high occurrence of fruits and seeds in spring. We did not find a significant difference in diet composition within sex and age, while an evident divergence was found between periods and sites. In particular, we noticed a decrease in prey abundance and in food-niche breadth from pre-reproductive period to post-reproductive period. Moreover a very clear difference was found between a site with allochthonous fishes and a site fish-free, with a more abundant and wide diet spectrum in the last one, as a result of the increased availability of prey. We highlighted the importance to take any possible actions to avoid the spread of fishes in other basins and to study the indirect impact of Procambarus clarkii, as possible vector of harmful trace element.