This paper presents new results of stable isotope analysis made on human and animal bones from Mesolithic–Neolithic sites (9500–5200 cal BC) in the Central Balkans. It reconstructs dietary practices in the Mesolithic and documents the development of new subsistence strategies and regional differences during the process of Neolithisation. We achieved these insights into dietary changes by analysing bone collagen δ13C (n = 75), δ15N (n = 75) and δ34S (n = 96) and comparing stable isotope data of Mesolithic–Neolithic communities from the Danube Gorges with the data of the first farmers who lived outside of the Gorges in the Central Balkans. The Bayesian model was employed to evaluate the relative importance of different animal proteins in human diet. Results bring a new overview and highlight important chronological and regional differences. They suggest that Late Mesolithic humans included more anadromous and potamodromous fish in their diet, which is consistent with archaeozoological evidence. On the other hand, differing from archaeozoological data, the model also points to a greater reliance on terrestrial carnivores (dogs) in the Late Mesolithic diet, a pattern that can be also explained by other dietary and environmental factors. In the Transitional and Neolithic period in the Gorges, some individuals have consumed fewer aquatic resources and favoured more terrestrial products. However, one site in the Gorges represents an exception—Ajmana, where we have the earliest farmers in this region since their subsistence economy was mainly oriented toward terrestrial products. Furthermore, results shows that Neolithic individuals inhumated at sites outside of the Danube Gorges in the Balkans had dietary patterns that vary in both terrestrial and freshwater resources, indicating that early farming communities had a diversified diet linked to a local natural environment. Comparative data finally indicates regional differentiations associated with locally available resources but also related to the traditions of prehistoric communities and to specific economic innovations.