Molecular technologies, such as metabarcoding, have become powerful tools for conservation purposes. Here, we present a non-invasive study analyzing the diet of one population of European pond turtle (Emys orbicularis) during its whole activity period and of four other populations during the same period, based on faecal sample, and using for the first time on this species, a long metabarcoding approach. Emys orbicularis is an emblematic freshwater species of wetlands in Europe. In several countries, this species is endangered and, in Switzerland, Emys orbicularis is ranked as critically endangered on the Swiss Red List. A national conservation program was created to reintroduce this species and raised the question if this reintroduced species could be a threat for other endangered species. We developed a new method of long metabarcoding analysis, using universal PCR primers to determine prey species occurrence in the faeces. The analysis conducted on 174 faeces collected on 142 individuals revealed 1153 preys from 270 species. Emys orbicularis consumed plants throughout the year with a more diverse diet during the reproduction period (April–June). This study therefore not only determines precisely the omnivorous and opportunistic diet of the Emys orbicularis, but also shows that this species is not a threat to its environment, as 85.5% of the consumed species were not list on the Swiss Red List. Moreover, it also demonstrated that the genetic analyses of faeces could be an efficient tool to determine trophic interaction with a high level of precision, yielding promising perspectives for food web ecology.