Cephalopods are an important component of Southern Ocean food webs but studies analysing their habitat and trophic ecology are scarce. Here, we use the Antarctic toothfish Dissostichus mawsoni as a biological sampler of Southern Ocean's cephalopods in the Ross, Amundsen, and D'Urville Seas. Ten cephalopod taxa were identified in the diet of the Antarctic toothfish, with Pareledone turqueti and Moroteuthopsis longimana being the only species present in all the three studied areas. DNA analysis conducted on squid flesh samples allowed identification of eight and two specimens of Mesonychoteuthis hamiltoni and M. longimana, respectively, proving this technique as a potential tool to improve the knowledge of cephalopods biodiversity and biogeography in the Southern Ocean. Stable isotopes were used to compare the habitat (δ13C) and trophic ecology (δ15N) between two life-stages of the two most abundant squid species (M. longimana and Psychroteuthis glacialis) from the D'Urville Sea (both squid species) and Amundsen Sea (only P. glacialis). Higher δ13C values in M. longimana suggest that this species inhabits waters near the Antarctic Polar Front, with incursions into sub-Antarctic waters, whilst P. glacialis spends its entire life in Antarctic waters. The most recently deposited part of the beak is enriched in 15N suggesting an increase in trophic level during squid growth. These results give us the first insights into the bathyal distribution of cephalopods in the Amundsen and D'Urville Seas, as well as into the ontogenetic changes of two of the most consumed squid species by top predators in this region. Such results are an important step towards improving the biogeography of Antarctic cephalopods, being of utmost importance to understand the biodiversity, food web structure, and functioning of this region.