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Abstract Animals must invest some portion of their metabolism to activities related to physiological maintenance and the remainder to processes related to the production of new biomass for growth and reproduction. Animal metabolism is fuelled by food, and the quality and quantity of food, along with the effort invested to obtain it, are fundamental to supporting populations. Biofilms are a primary basal food resource within riverine food webs, and it is thought that their nutritional value for animals decreases with age due to dynamic changes in community composition. We sought to test assumptions of spatiotemporal changes to biofilm nutritional value by assessing variations in biofilm mass and fatty acid composition in three rivers for 73 days. We also used a multi‐prong eDNA approach to characterize changes to biofilm fungal (ITS1–4), bacterial (16S), and algal (23S) community compositions. We anticipated biofilm food value to decrease with biofilm age due to shifts in composition from high‐quality green algae and diatoms to low‐quality cyanobacteria and filamentous algae. Our results partially support this contention; biofilm food value, assessed as a combination of fatty acid mass per unit area (in grams per square meter) and concentration of fatty acids (in milligrams per gram), was dynamic and peaked between 24 and 43 days following submersion. After 43 days, biofilm food value decreased. However, despite significant temporal changes in biofilm community composition and a decrease in overall lipid concentration, the proportions of different fatty acid classes among total lipids did not vary. Instead, the observed increase in the abundance of cyanobacteria and filamentous algae compared with diatoms and green algae, along with higher quantities of lipid‐poor extracellular polymeric substances (EPS), likely contributed to the reduction in overall lipid concentration relative to the biofilm dry mass. Here we present a novel approach to balance consumer energetic costs with food quality within aquatic food webs. Our results have important implications for river management and provide valuable information for the use of environmental water to support lotic ecosystems.