Understanding ecological relationships between humans and marine predators is challenging, but crucial for the implementation of sustainable management practices. Comprehensive estimation of pinniped diet is essential for assessing their interaction with commercial fisheries as well as to increase knowledge on ecological interactions and food webs. In Iceland, salmonid fisheries are economically important and harbour seals are often culled in the vicinity of salmon estuaries to prevent potential seal predation on salmonid stocks. There is a lack of information on harbour seal diet, and due to uncertainty of the accuracy of methods traditionally used to estimate harbour seal diet (e.g. hard-part analysis of faeces) it is necessary to improve analysis methods. In this study, we investigated the diet of harbour seals hauling out in an estuary area in north western Iceland between June and September of 2010 and 2011 by DNA analysis of prey in faeces using DNA barcoding. The results were compared to previously published results from morphological analysis. Our results showed that harbour seals mainly consumed sand eels, flatfishes, gadoids, herring and capelin. Species consumed by the seals were at large in agreement with species availability in the area. The results from molecular and morphological analysis were similar in regards of important prey species. However the species diversity was lower in the morphological analysis and 37.5% of the samples included prey items that were unidentifiable in the morphological analysis. Notably, despite salmon, trout and char availability in the study area, neither of the methods found evidence of salmonids in the harbour seal diet. Since the main reason for culling harbour seals in Iceland is to reduce predation on salmonids, the finding that salmonids are not important prey species is important in terms of management implications, especially in the light of a recent severe decline in the Icelandic harbour seal population.