Landuse change alters the quantity and supply of stream energy resources from allochthonous (catchment-derived) to autochthonous (stream-derived) sources. This alteration has prompted the need for better understanding of C and N pathways through stream food webs. We added stable ¹⁵N-NH₄ and ¹³C-acetate isotopes as tracers to experimental stream channels containing 1 of 4 key basal food resources: autotrophic epilithon, heterotrophic epilithon, fine particulate organic matter (FPOM), and coarse particulate organic matter (CPOM). Our goal was to evaluate the importance of these foods to 2 scrapers (Deleatidium sp. [mayfly] and Potamopyrgus antipodarum [snail]) and a facultative shredder (Olinga feredayi [caddisfly]). A 5^th potential food source, new heterotrophic biofilm, grew on all surfaces during the course of the experiment. Isotope levels in the food resources and the invertebrates were measured weekly during the 3-wk isotope addition and for 2 wk after the addition. Changes in nutrient concentrations between the channel inflows and outflows provided measures of instream net uptake. Net uptake of P by autotrophic epilithon (mean = 0.09–0.24 mg dissolved reactive P [DRP] m⁻² h⁻¹) was 3× higher than uptake by other basal resources. Autotrophic epilithon removed substantial quantities of dissolved N, predominantly as NO₃-N (2.1–9.3 mg NO₃-N m⁻² h⁻¹), whereas the other basal resource types removed only NH₄-N at a rate that was 2 orders of magnitude slower (mean = 0.04–0.08 mg NH₄-N m⁻² h⁻¹) than uptake by autotrophic epilithon. NH₄-N and dissolved organic C isotope tracers were taken up predominantly by heterotrophic epilithon and new biofilms. We developed a dynamic mixing model to separate the contribution to the invertebrates of each treatment food source relative to the new heterotrophic biolfilm. Mayflies obtained almost 100% of their body C and N from new biofilm except when offered autotrophic epilithon, which contributed >50% to their diets. Olinga and Potamopyrgus obtained most of their C and N from the treatment food sources, but in the particulate organic matter treatments (FPOM and CPOM) the contribution of new biofilm was much larger (∼10–60%). Our results suggest that heterotrophic biofilms are a significant component of stream food webs.