Non-invasive methodological approaches are highly recommended and commonly used to study the feeding ecology of elusive and threatened mammals. In this study, we use multiple lines of evidence to assess the feeding strategies of the endangered Southern river otter, by determining seasonal prey availability (electrofishing), analysis of undigested prey remains (spraints), and the use of stable isotopes (δ(15)N and δ(13)C) in otter spraints (n = 262) and prey in a wetland ecosystem of southern Chile (39°49'S, 73°15'W). Fecal and isotopic analyses suggest that the otter diet is restricted to a few prey items, particularly the less-mobile, bottom-living, and larger prey such as crayfish (Samastacus spinifrons, 86.11%) and crabs (Aegla spp., 32.45%), supplemented opportunistically by cyprinids (Cyprinus carpio, 9.55%) and catfish (Diplomystes camposensis, 5.66%). The results suggest that the river otter is highly specialized in bottom foraging. Isotopic signatures of food sources and feces revealed a mid-upper trophic position for the Southern river otter, with either higher or lower δ(15)N values than their potential prey items. δ(13)C values for river otters were less enriched than their potential food resources. We suggest that due to their narrow trophic niche and possible dependence on only a few food items, this species may be highly vulnerable to the reduction in its prey populations. Finally, maintaining the ecological interactions between Southern river otters and their prey is considered a central priority for the survival of this endangered carnivore mammal.