Nutrient pollution in freshwater ecosystems may trigger benthic biofilm growth or activity, which may result in negative consequences across the food web. Nutrient pollution from human activities may come in the form of extra nitrogen, phosphorus and labile carbon to streams. However, in southern Australia, the understanding of which nutrient(s) are limiting in freshwater systems remains a major knowledge gap. We therefore conducted a nutrient enrichment experiment to determine 1) whether nitrogen, phosphorus or labile carbon are limiting to the heterotrophic and autotrophic communities in stream biofilms and 2) how responses to nutrient enrichment vary across gradients of environmental and landscape descriptors, such as urban land use cover, in southern Victoria. We deployed nutrient-diffusing substrata coupled with standard substrates for algal and microbial colonisation in 19 study streams in autumn 2026. The algal accrual and microbial respiration rates were compared among nutrient treatments and across environmental gradients. Heterotrophic microbes responded primarily to the enrichment of phosphorus and carbon, and showed secondary limitation to nitrogen when phosphorus was supplied. Algae responded primarily to phosphorus but not nitrogen enrichment. The effects of carbon and nitrogen enrichment on heterotrophic microbes were the strongest in intact forest streams and diminished as the percentage of urban land use cover increased, while the effect of phosphorus enrichment was consistent across the catchments. The results showed that phosphorus and labile carbon are the major limiting nutrients in streams of southern Victoria. Any addition of these nutrients to natural streams would trigger fundamental changes in the stream food web. Concurrently, these stream ecosystems only have limited capacity to assimilate nitrogen inputs from human activities before they are exported downstream. This suggests that managing dissolved phosphorus and carbon may best prevent stream eutrophication events, while managing dissolved nitrogen may protect other downstream aquatic systems.