The Ross Sea, Antarctica, supports large populations of Emperor Penguin (Aptenodytes forsteri) and Adélie Penguin (Pygoscelis adeliae), two key meso-predators that occupy high trophic levels. Despite these species are largely studied, little is known about their diet outside the breeding period. In the present study, we investigated the intra-annual diet of Adélie and Emperor Penguins belonging to five colonies in the Ross Sea through the stable isotope analysis of different tissues (feathers and shell membranes), synthetized in different seasons, and guano that indicates recent diet. Penguin samples and prey (krill and fish) were collected during the Antarctic spring–summer. δ13C and δ15N of tissues and guano indicate spatio-temporal variation in the penguin diet. The krill consumption by Adélie Penguins was lowest in winter except in the northernmost colony, where it was always very high. It peaked in spring and remained prevalent in summer. The greatest krill contribution to Emperor Penguin’s diet occurred in summer. The relative krill and fish consumption by both species changed in relation to the prey availability, which is influenced by seasonal sea ice dynamics, and according to the penguin life cycle phases. The results highlight a strong trophic plasticity in the Adélie Penguin, whose dietary variability has been already recognized, and in the Emperor Penguin, which had not previously reported. Our findings can help understand how these species might react to resource variation due to climate change or anthropogenic overexploitation. Furthermore, data provides useful basis for future comparisons in the Ross Sea MPA and for planning conservation actions.