Abstract Algae are generally a high‐quality diet source because they provide essential compounds to aquatic consumers. In forested stream ecosystems, the availability of high‐quality algae is low compared to terrestrial organic matter, which may constrain the dietary transfer of essential compounds to consumers. However, there could be other overlooked high‐quality resource pools that provide essential compounds to consumers in river ecosystems. We conducted a field study along a subalpine river continuum in Austria to identify the nutritional role of a ‘hidden’ food resource for aquatic consumers; the biofilms growing on the underside of rocks (dark biofilms). Dark and light (i.e. upper surface of rocks) biofilms and invertebrates were collected, and their fatty acid (FA) composition was analysed. Compared with light biofilms, dark biofilms contained greater proportions of bacterial FA, long‐chain saturated FA (biomarkers of terrestrial plants) and oleic acid (18:1ω9; a fungal biomarker), but a lower proportion of algal FA, especially omega‐3 polyunsaturated FA (ω3 PUFA). The ω3 PUFA composition in dark biofilms was strongly correlated with that in light biofilms. Furthermore, the overall FA profiles of dark biofilms were significantly associated with invertebrate FA profiles. Strong correlations were also observed between invertebrates and dark biofilms for bacterial FA and the ω3 PUFA eicosapentaenoic acid (EPA, 20:5ω3). Synthesis. This field study demonstrates that dark biofilms are a high‐quality resource pool for invertebrates in river ecosystems that is often overlooked. Similar to light biofilms, dark biofilms provide physiologically important FA and bacterial FA for stream invertebrates. However, these high‐quality resources are threatened by increasing human disturbances to river ecosystems. Future research is required to better understand how the quality of both dark and light biofilms changes in response to human disturbance, and how this affects dietary energy transfer to upper trophic consumers, including fish and humans.