Animal metabolism is fuelled by food, and the quality and quantity of food, along with the effort invested to obtain it, is fundamental to supporting populations. Biofilms are a primary basal food resource within rivers, and it is thought that their nutritional value for animals decreases with age. We sought to test assumptions of spatio–temporal changes to biofilm taxa composition and subsequent implications for food value in lowland rivers. We assessed biofilm biotic composition, mass, and fatty acid profiles on submerged wooden blocks on floating platforms in three rivers for 73 days. We used a multi-prong eDNA approach, sequencing regions of the ITS1–4 gene (fungi), 16S (bacteria) and 23S (algae) ribosomal RNA (rRNA) genes to examine the structure of biotic communities with time. We anticipated that biofilm food value for consumers would decrease with biofilm age due to changes in composition. Our results support this contention in part; biofilm food value, assessed as a combination of areal mass of high–quality fatty acids (g m-2) and the mass of high–quality fatty acids relative to biofilm dry weight (mg g-1), was dynamic and peaked between 24– and 43–days following submersion. After 43–days, biofilm food value and availability decreased. However, the primary driving mechanism differed from what we expected. Despite significant changes to biofilm species composition and a reduction in total lipids per unit mass of biofilm, the proportions of different fatty acids among total lipids did not vary. Our results suggest biofilm food quality in lowland rivers is driven by three factors; 1/the ongoing addition of lipid–poor detritus from the water column 2/ a shift from diatom and green algae dominated biofilms to a domination by cyanobacteria and filamentous algae and 3/ a decrease in the ratio of biofilm cell bodies to extracellular polymeric substances.