Ecologists traditionally investigate the feeding ecology of vertebrates by visual identification of prey in their scats using a microscope. This analysis, however, presents some pitfalls, such as poor prey identification at genus/species level. DNA barcoding is an alternative to the traditional diet analysis, recently applied to investigate the diet of several vertebrates. It allows the identification of cryptic or not-recognizable prey portions basing their identification on the analysis of conserved DNA fragments. Here, we combined microscope analysis with DNA barcoding to investigate the diet of the Eurasian otter (Lutra lutra) in an area recently recolonized by the species, in the surroundings of Tarvisio, NE Italy. We collected 102 spraints and analysed all of them at microscope. A random subsample (N = 50) was then analysed through DNA barcoding, via amplification of the V9 region of the SSU 18S-rDNA 18S gene. Furthermore, we assessed seasonal variation in otter diet and whether otters select certain salmonids size classes, to identify potential conflict with anglers. According to the microscope analysis, otters ate primarily fish (salmonids and European bullhead), secondarily amphibians and, more rarely, crayfish, both annually and seasonally. DNA barcoding analysis confirmed the microscope results, attributing some salmonids sequences to the brown trout (Salmo trutta). The lack of potential prey species reference sequences in the GenBank database hampered us in identifying the other taxa found in the spraints at more informative levels. Consumed salmonids and bullheads were of small size. In particular, otters selected salmonids size class of 100 mm, which is much smaller than the minimum catchable size for angling. Overall, our study confirms DNA barcoding potential for otter diet analyses, highlighting the need of additional markers to improve molecular prey identification and suggesting that otters do not constitute a significant threat to anglers in the area.