Benthic biofilms have been identified using stable isotope analysis (SIA) as an important resource supporting many freshwater food webs. However, biofilm d 13 C signatures are highly variable in freshwaters, which may hamper our understanding of energy flow through food webs in these systems. There has been little consideration of the influence that substratum may have on biofilm d 13 C signature variability and energy flows to primary consumers. We investigated the effect of organic and inorganic substrata on biofilm dynamics by examining: (1) temporal variability of biofilm stable isotope (d 13 C, d 15 N) signatures on allochthonous leaf-litter (Eucalyptus camaldulensis) and cobble substrata over 12 months in a lowland river in south-eastern Australia; and (2) the effect of substrata on biofilm energy flows to a grazer snail, Physa acuta (Gastropoda: Physidae), using SIA and ecological stoichiometry in a laboratory experiment. The temporal study indicated that cobble biofilm varied significantly in d 13 C signature during the 12 months (up to 11%), whereas the d 13 C signature of leaf biofilm was less variable (less than 2%). In contrast, biofilm d 15 N signatures varied temporally on both cobble (2.6%) and leaf (1%) substrata. This suggests that leaf biofilm was more reliant on leaf tissue for carbon and therefore less limited by carbon supply than cobble biofilm whereas for nitrogen biofilm on both substrata was reliant on external sources. In the laboratory experiment, snails fed leaf biofilm reflected more of an allochthonous d 13 C signature than cobble biofilm fed snails, suggesting assimilation of leaf carbon via the heterotrophic microbial community within the biofilm. Snails grew largest on cobble biofilm, which had lower C:N ratios than leaf biofilm. Our results demonstrate that the type of substratum can influence the temporal variability of biofilm d 13 C signatures and energy flow to primary consumers.