We studied the fatty acid (FA) content and composition of ten zoobenthic species of several taxonomic groups from different freshwater bodies. Special attention was paid to essential polyunsaturated fatty acids, eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3), and arachidonic acid (ARA, 20:4n-6); and the n-3/n-6 and DHA/ARA ratios, which are important for consumers of higher trophic levels, i.e., fish. The content and ratios of these FA varied significantly in the studied zoobenthic species, consequently, the invertebrates were of different nutritional quality for fish. Eulimnogammarus viridis (Crustacea) and Dendrocoelopsis sp. (Turbellaria) had the highest nutrition value for fish concerning the content of EPA and DHA and n-3/n-6 and DHA/ARA ratios. Using canonical correspondence analysis we compared the FA profiles of species of the studied taxa taking into account their feeding strategies and habitats. We gained evidence that feeding strategy is of importance to determine fatty acid profiles of zoobenthic species. However, the phylogenetic position of the zoobenthic species is also responsible and may result in a similar fatty acid composition even if species or populations inhabit different water bodies or have different feeding strategies.