Total lipid content of eight Greek freshwater fish species examined [bream (Abramis brema), carp (Cyprinus carpio), chub (Leuciscus cephalus), crucian carp (Carassius carassius), ide (Leuciscus idus), nase (Chondrostoma nasus), pike-perch (Lucioperca lucioperca), and sheatfish (Silurus glanis)] ranged between 0.6 and 3.5%. The most abundant fatty acids were palmitic (16:0), palmitoleic (16:1), oleic (18:1), eicosapentaenoic (20:5) and docosahexaenoic (22:6). Palmitic acid was the dominant saturated fatty acid averaging ca. 56% of total saturated acids. Oleic acid (18:1) was the most abundant monoenoic fatty acid. The level of total ω-3 polyunsaturated fatty acids (PUFA) was higher than the total ω-6 fatty acid, ranging from 12.4 to 31.8%, while the ratios ranged between 1.2 and 2.9. The major ω-3 PUFAs were 20:50 (6.0–11.8%) and 22:6ω3 (4.0–15.3%) and the major ω-6 PUFA was 18:2ω6 (2.8–8.0%), followed by 20:4ω6 (0.8–3.8%). These data revealed that freshwater fish were good sources of eicosapentaenoic and docosahexaenoic acids.