The Pyrenean desman (Galemys pyrenaicus) is a small endangered semi-aquatic mammal endemic to the Pyrenean Mountains and to the northern half of the Iberian Peninsula whose ecology and biology are still poorly known. The aim of this study was to identify Pyrenean desman faeces and to analyze its diet from this material using next-generation sequencing methods. We amplified and sequenced a small DNA minibarcode (133 bp) of the COI gene in twenty-four putative faeces samples of Pyrenean desman and successfully identified the species in 16 samples. Other identified species were mammals, birds and amphibians, evidencing the potential application of our methods to a larger panel of taxa. In the Pyrenean desman faeces, we were able to identify nineteen prey species with a positive match (more than 98% of identity with a reference sequence) and eleven putative prey species with lower identity scores (90–96%). The nineteen species belonged to four orders and eleven families among which Trichoptera and Hydropsychidae were the most frequent, respectively. Future improvements could be obtained by extending the reference DNA sequence collection to reach precise identifications over the Desman's range and by increasing the sampling to gain a better knowledge of the local diet of this endangered species. Such information is of great importance to propose the best management measures for its conservation.