Mosses can be responsible for up to 100% of net primary production in arctic and subarctic tundra, and their associations with diazotrophic cyanobacteria have an important role in increasing nitrogen (N) availability in these pristine ecosystems. Predictions about the consequences of climate change in subarctic environments point to increased N mineralization in soil and higher litter deposition due to warming. It is not clear yet how these indirect climate change effects impact moss-cyanobacteria associations and N2 fixation. This work aimed to evaluate the effects of increased N and litter input on biological N2 fixation rates associated with the feathermoss Hylocomium splendens from a tundra heath. H. splendens samples were collected near Abisko, northern Sweden, from a field experiment with annual additions of ammonium chloride and dried birch litter and the combination of both for three years. Samples were analyzed for N2 fixation, cyanobacterial colonization, C and N content and pH. Despite the high N additions, no significant differences in moss N content were found. However, differences between treatments were observed in N2 fixation rates, cyanobacterial colonization and pH, with the combined ammonium+litter treatment causing a significant reduction in the number of branch-colonizing cyanobacteria and N2 fixation, and ammonium additions significantly lowering moss pH. A significant, positive relationship was found between N2 fixation rates, moss colonization by cyanobacteria and pH levels, showing a clear drop in N2 fixation rates at lower pH levels even if larger cyanobacterial populations were present. These results suggest that increased N availability and litter deposition resulting from climate change not only interferes with N2 fixation directly, but also acidifies moss microhabitats and reduces the abundance of associated cyanobacteria, which could eventually impact the N cycle in the Subarctic.