Abstract There is growing recognition of the importance of food quality over quantity for aquatic consumers. In streams and rivers, most previous studies considered this primarily in terms of the quality of terrestrial leaf litter and importance of microbial conditioning. However, many recent studies suggest that algae are a more nutritional food source for riverine consumers than leaf litter. To date, few studies have quantified longitudinal shifts in the nutritional quality of basal food resources in river ecosystems and how these may affect consumers. We conducted a field investigation in a subalpine river ecosystem in Austria to investigate longitudinal variations in diet quality of basal food sources (submerged leaves and periphyton) and diet source dependence of stream consumers (invertebrate grazers, shredders, filterers and predators, and fish). Fatty acid (FA) profiles of basal food sources and their consumers were measured. Our results indicate systematic differences between the FA profiles of terrestrial leaves and aquatic biota, that is periphyton, invertebrates and fish. Submerged leaves contained very low proportions of long‐chain polyunsaturated fatty acids (LC‐PUFAs), which were conversely rich in aquatic biota. While the FA composition of submerged leaves remained similar among sites, the LC‐PUFAs of periphyton increased longitudinally, which was associated with increasing nutrients from upstream to downstream. Longitudinal variations in periphyton LC‐PUFAs were reflected in the LC‐PUFAs of invertebrate grazers and shredders, and further tracked by invertebrate predators and fish. However, brown trout Salmo trutta contained a large proportion of docosahexaenoic acid (DHA, 22:6ω3), a LC‐PUFA almost entirely missing in basal sources and invertebrates. The fish accumulated eicosapentaenoic acid (EPA, 20:5ω3) from invertebrate prey and may use this FA to synthesize DHA. Our results provide a nutritional perspective for river food web studies, emphasizing the importance of algal resources to consumer somatic growth and the need to account for the longitudinal shifts in the quality of these basal resources.