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The Antarctic toothfish (Dissostichus mawsoni) is the largest notothenioid species in the Southern Ocean, playing a keystone role in the trophic web as a food source for marine mammals and a top predator in deep-sea ecosystems. Most ecological knowledge on this species relies on samples from areas where direct fishing is allowed, whereas in areas closed to fishing, such as the Antarctic Peninsula (AP), there are still key ecological gaps to ensure effective conservation, especially regarding our understanding of its trophic relationships within the ecosystem. Here, we present the first comprehensive study of the feeding behavior of Antarctic toothfish caught in the northern tip of the AP, during two consecutive fishing seasons (2019/20 and 2020/21). Stomach content was analyzed according to size-classes, sex and season. Macroscopic morphological analysis was used to identify prey, whereas DNA analysis was used in highly digested prey items. Fatty acid analysis was conducted to determine the prey’s nutritional composition. The diet mainly consisted of Macrouridae, Cephalopoda, Anotopteridae, and Channichthyidae. Other prey items found were crustaceans and penguin remains; however, these were rare in terms of their presence in stomach samples. Sex had no effect on diet, whereas size-class and fishing season influenced prey composition. From 27 fatty acid profiles identified, we observed two different prey groups of fishes (integrated by families Anotopteridae, Macrouridae and Channichthyidae) and cephalopods. Our results revealed a narrow range of prey items typical of a generalist predator, which probably consumes the most abundant prey. Understanding the diet and trophic relationships of Antarctic toothfish is critical for a better comprehension of its role in the benthic-demersal ecosystem of the AP, key for ecosystemic fisheries management, and relevant for understanding and predicting the effect of climate change on this species.