To identify the major prey items for Antarctic toothfish (Dissostichus mawsoni) in the small-scale research unit (SSRU) 5841C in the east Antarctic, their stomach contents, fatty acid (FA) compositions and stable carbon and nitrogen isotope ratios were determined and compared with those of species caught as by-catch and collected from toothfish stomachs. Stomach content analyses showed that Antarctic toothfish fed primarily on fish and to a lesser extent squid. FA profiles in muscle tissues of Antarctic toothfish were very similar to those of Channichthyidae caught as by-catch and several species collected from toothfish stomachs, including unidentified icefish, Arctozenus risso, Macrourus spp., and Gymnodraco acuticeps, indicating a trophic connection between them. δ¹⁵N values of Antarctic toothfish were higher than for the other species collected, indicating a higher trophic position. This is the first study to provide information on the diet of Antarctic toothfish and the trophic relationship between the toothfish and other species in the east Antarctic using these methods. Further studies on the trophic relationship between Antarctic toothfish and other species and a regional comparison of their dietary composition by the collection and subsequent biomarker analyses of more species is needed to understand better the carbon flow through Antarctic ecosystems.