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The main objective of this paper is to assess paleodiets and subsistence patterns in hunter-gatherer groups of the eastern Pampa-Patagonia transition during the Middle Holocene (ca. 6000–4100years BP). Using a multivariate diet reconstruction model that incorporates δ13Cap, δ13Ccol, and δ15N holistically, human palaeodiets were characterized through the identification of dietary patterns relative to the consumption of terrestrial (e.g., vegetal and animal) and marine resources (e.g., fish). Thus, both spatial and temporal variations in diet and subsistence, as well as issues related to mobility and the use of landscape (coast-inland), were explored. Nine adult individuals of both sexes from four archeological sites were analyzed. The results of δ13Ccoland δ15N for the study area indicated that the individuals buried close to the Atlantic coast (Tres Bonetes 1-colección Donnay and Cantera de Rodados Villalonga sites) exhibited similar values among them, which differed statistically from those individuals buried in sites located inland (Loma de Los Morteros and La Modesta). While the first group of individuals showed a range of marine to mixed diets, the second group was characterized by terrestrial values. The observed differences were interpreted as the product of possible territorial demarcations. For the study area, Middle Holocene dietary trends were also compared with those of the Late Holocene, which indicated temporal variations in the diet between ca. 6000–400years BP. Finally, at the macro-regional level, the results obtained here showed certain differences and similarities from those of northeastern Patagonia and southeastern Pampa. Such macro-regional trends in paleodiets are also discussed here.