Fish, benthic macroinvertebrates, macrophytes, biofilm, plankton, and terrestrial material were analyzed for stable nitrogen (δ15N) and carbon isotopes (δ13C) to study the food web structure of a double-basined, ultra-oligotrophic lake in northwest Patagonia. The δ15N values of all biota were consistently higher in Moreno East basin, probably caused by a higher anthropogenic impact; however, the estimated trophic positions of fish showed no differences between basins, indicating that fish are feeding from food chains with different δ15N baselines. Fish δ13C values were higher in Moreno West basin but no pattern was observed for the rest of the organisms analyzed. The different hydrogeomorphic characteristics between the basins could lead to these differences in the C signatures of fish. SIAR (Stable Isotope Analysis in R) mixing model was used to study possible food sources for adult fish and the results were contrasted with published gut content analysis. According to the model, in both basins small size fish such as Small Puyen (Galaxias maculatus) and juvenile fish, had the highest relative isotopic contribution to introduced Rainbow Trout (Oncorhynchus mykiss) and Brook Trout (Salvelinus fontinalis), which had lower contributions from other sources such as insect larvae, decapods, and snails. On the contrary, decapods and insect larvae were isotopically more important for the native Creole Perch (Percichthys trucha) than small fish in both basins. An ontogenetic shift in the carbon source and trophic level was observed in Puyen Chico and Rainbow Trout from the West basin.