The study of fatty acid biomarkers in trophic structures at sub-polar latitudes is fundamental in describing energy fluxes across ecosystems characterized by complex inter-specific interactions. Due to the presence of certain essential fatty acids obtained exclusively from predator–prey interactions, fatty acid biomarkers are widely used to identify trophic interactions. This study analyzed fatty acid compositions in three species inhabiting a relatively pristine Patagonian fjord. This fjord is geographically difficult to access, so there are very little sampling opportunities, biological and oceanographic information. In the three species collected (Ctenodiscus australis (Loven in Lütken 1871) (Echinodermata, Asteroidea, Ctenodiscidae); Munida gregaria (Fabricius 1793) (Arthropoda, Malacostraca, Munididae); Eleginops maclovinus (Cuvier 1830) (Chordata, Actinopterygii, Eleginopsidae)) along this remote area were evaluated their fatty acid trophic markers as a tool to differentiate dietary components and dietary habits. The study reported significant differences in the amount of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA), with the highest concentrations of all fatty acids in M. gregaria. The last suggests that M. gregaria is considered as a good quality food source or biological component that might support the fjord trophic web in the Southern Hemisphere. The results describe diet compositions in sampled species, and differences among species for fatty acid compositions and proportions. This provides an initial basis for future modeling or projecting how benthic ecosystems of fjords and Patagonian channels respond to food intake, particularly in environments associated with glacial systems characterized by a low phytoplankton biomass and greater sensitivity to climate variability.