Wild boar and roe deer samples from an excavation in Dispilio, Greece, were subjected to collagen extraction protocols to reconstruct the paleoecological regime. Radiocarbon (14C) analysis suggested the Middle/Late Neolithic period and the database was updated with collagen samples. The 14C model concluded to a possible local deforestation effect in the settlement subbasin confirmed by sediment δ13C and δ15N values. Carbon isotope values in collagen samples concluded in C3 plant type. Both carbon and nitrogen isotopes indicated the differences in dietary habits and/or metabolic system between the two Late Neolithic I species. Roe deer samples were classified as purely herbivorous. δ15N values of wild boar collagen samples from Dispilio reflected a diet mainly characterized by terrestrial protein. Compared to literature data, wild boar samples from the Dispilio excavation concluded that the animals might have lived close to the settlement where their diet could be supplemented by a consistent animal protein fraction. Finally, it is concluded that rainfall is an important factor that affects plant, and consequently animal, δ15N values. Therefore, the rainfall regime should always be considered in paleodietary studies.