Our study is a first attempt to characterize seasonal fatty acid (FA) profiles of foodweb components in a small forested stream. We measured FA content of autochthonous food sources (aquatic primary producers = periphyton, green algae, red algae, bryophytes), allochthonous food resources (terrestrial matter = benthic and transported organic matter [BOM and TOM, respectively]), and macroinvertebrate consumers (Hydropsyche spp., Ephemerella spp., isopods, oligochaetes). We examined whether FAs could be used as trophic markers and tried to identify which food sources best provided macroinvertebrates with essential FAs (EFA, ω3 and ω6 groups). Primary producers consistently had greater content of several EFAs (18:2ω6 and 18:3ω3 in green algae, 20:5ω3 in diatoms, 20:4ω6 in bryophytes) than did terrestrial matter. The ratio of Σω3/Σω6 FAs, a putative marker of the relative amount of autochthonous vs allochthonous matter, was greatest in macroinvertebrates, followed by algae, and was significantly correlated with chlorophyll a content of food sources (periphyton, ultrafine BOM, and TOM). The seasonal dynamics of EFA content of BOM and TOM varied with particle size. Other FAs were identified as specific markers for diatoms (20:5ω3 [eicosapentaenoic acid], 16:1ω7, 16:ω4s, 16C-polyunsaturated FAa [PUFAa]), green algae (18:3ω3 [α-linolenic acid], 18:2ω6 [linoleic acid], 16C-PUFAb), and bryophytes (20:4ω6, 20:3ω3). Terrestrial matter had higher levels of bacterial and fungal FAs than did primary producers. Nonmetric multidimensional scaling analyses based on FA spectra of foodweb components in early spring (open canopy) and mid-summer (shaded canopy) confirmed that Ephemerella and Hydropsyche consumed mainly autochthonous food sources, even during the shaded summer period. Isopods and oligochaetes consumed a mixed diet of terrestrial matter and algae. Autochthonous food sources may be a more important part of the diets of benthic macroinvertebrates in forested streams than previously recognized.