Ecological information regarding trophic patterns and the inherent structure of freshwater aquatic communities is considered fundamental in ecological research. In this study, the trophic patterns of the Greek freshwater fish fauna, belonging to the Balkan biodiversity hotspot, were investigated, and comparisons of freshwater fish species feeding habits among the Greek freshwater ecoregions were performed. The analyses based on the widely used trophic level index and the available composition feeding datasets, along with the utilization of clustering methods, indicated seven major distinct trophic guilds in the Greek ecoregions. The trophic level of the studied fish species ranged from 2.0 to 4.5, thus being within the expected values for freshwater ecosystems, revealing the presence of both top predators and primary consumers. The highest trophic level values were found in the ecoregions of northern Greece. The results also exhibited considerable predominance of higher-trophic-level zooplanktivorous and insectivorous freshwater fish species in the mainland, compared to lower-trophic-level opportunist species in the island-isolated ecoregions. These results could be used for the application of ecosystem-based models and the formulation of conservation and fishery management schemes.