Through application of carbon (C) and nitrogen (N) stable isotope analyses, we investigated the benthic trophic structure of the upper-slope off southern Chile (~45°S) including a recent methane seep area discovered as part of this study. The observed fauna comprised 53 invertebrates and seven fish taxa, including remains of chemosymbiotic fauna (e.g. chemosymbiotic bivalves and siboglinid polychaetes), which are typical of methane seep environments. While in close-proximity to a seep, the heterotrophic fauna had a nutrition derived predominantly from photosynthetic sources (δ13C > –21‰). The absence of chemosynthesis-based nutrition in the consumers was likely a result of using an Agassiz trawl to sample the benthos, a method that is likely to collect a mix of fauna including individuals from adjacent non-seep bathyal environments. While four trophic levels were estimated for invertebrates, the fish assemblage was positioned within the third trophic level of the food web. Differences in corrected standard ellipse area (SEAC), which is a proxy of the isotopic niche width, yielded differences for the demersal fish Notophycis marginata (SEAC = 5.1‰) and Coelorinchus fasciatus (SEAC = 1.1‰), suggesting distinct trophic behaviours. No ontogenic changes were detected in C. fasciatus regarding food sources and trophic position. The present study contributes the first basic trophic data for the bathyal area off southern Chile, including the identification of a new methane seep area, among the furthest south ever discovered. Such information provides the basis for the proper sustainable management of the benthic environments present along the vast Chilean continental margin.