Abstract 1. This study evaluated how the availability of nutrients and organic carbon interact to influence the associations between autotrophic and heterotrophic microorganisms during lake biofilm development. Considering that decomposers are often better competitors for nutrients than producers in aquatic environments, we hypothesized that heterotrophs would outcompete autotrophs for available nutrients unless heterotrophs were limited by organic carbon provided by autotrophs. 2. To test our hypothesis, we evaluated autotrophic (algae) and heterotrophic (fungi, bacteria) biomass in response to a factorial enrichment of nutrients (nitrogen and phosphorus in combination) and glucose using nutrient diffusing substrates with either inorganic or organic discs in a subalpine lake. In the field, nutrient diffusing substrates were exposed to either natural sunlight or placed under a darkened experimental canopy to evaluate the response of heterotrophs to nutrients and carbon subsidies in the presence or absence of algae. We expected that heterotrophs would be limited by organic carbon on inorganic substrates in the absence of autotrophic production (i.e., dark treatments), and that organic substrates would provide a carbon subsidy for heterotrophic metabolism. 3. Fungi were stimulated by nutrient enrichment on inorganic substrates in the presence of algae (light treatment), but not in the dark (without algae). The response of fungi to algal presence on inorganic substrates was similar in magnitude to the response of fungi to nutrients and glucose substrates incubated in the dark. In contrast to our expectations, elevated algal biomass did not stimulate heterotrophic bacteria in the presence of elevated nutrient levels on inorganic substrates, possibly owing to antagonistic interactions between bacteria and fungi. 4. The positive effect of nutrients on algal biomass was significantly reduced in favor of heterotrophs when nutrients were combined with glucose, suggesting that heterotrophs were able to outcompete algae for available nutrients in the absence of carbon limitation. 5. Synthesis. These results expand our understanding of how the availability of limiting resources govern the outcomes of complex interactions among microorganisms in aquatic biofilms, and suggest that background levels of organic carbon regulate producer and decomposer responses to nutrient availability during biofilm development.