Abstract The fatty acid (FA) composition of spionids co-occurring in shallow-water and occupying various habitats was analyzed to characterize the spectrum and diversity of their food sources and elucidate their feeding habits and trophic relationships. These included free-living and tube-dwelling species, and also opportunistic and specialized shell-borers and their hosts. A plot of principal component analysis (PCA) scores based on the FA compositions of nine spionid species recognized five distinct groups with different feeding strategies and revealed differences not only between the species, but also within the same species from various habitats. The predominance of 20:5n-3, C16 polyunsaturated FAs, and 16:1n-7 in suspension feeders indicated that they feed on diatoms. Benthic dinoflagellates and diatoms, as well as bacteria, constituted a major part in the diet of deposit feeders. On the basis of high 22:6n-3 and 18:1n-9 contents, Dipolydora commensalis was attributed to omnivorous with a predominance of animal dietary input. The selective detritivore Polydora glycymerica relies on a microbial food web. FAs of Laonice aff. cirrata indicated the importance of foraminifera in the diet of this subsurface feeder. Polychaetes were found to have a capability of changing their feeding habits depending on habitat. Specialized shell-borers adapt their feeding habits to the host’s diet. The results obtained provide evidence that these species actually represent several non-overlapping groups characterized by different feeding strategies, which leads to weakening of their competitive relationships. Partitioning of food sources is regarded as one of the main mechanisms explaining the successful coexistence of spionid species.