Litter-associated fungi are key intermediaries in carbon flow and nutrient cycling in streams. They can obtain nitrogen (N) and phosphorus (P) from both substrates and the water column. We used streamside channels at the Coweeta Hydrologic Laboratory (NC, USA) to test the effects of dissolved inorganic N (DIN) and soluble reactive P (SRP) on fungal reproduction (sporulation rate of aquatic hyphomycetes) and community structure associated with plant litter (maple and rhododendron leaves and wood). To evaluate sporulation and fungal communities, we combined traditional microscopy with next-generation sequencing (NGS, Illumina) targeting fungal ITS2 rDNA region. Our bioinformatics pipeline included QIIME2 coupled with the UNITE database supplemented with >100 novel sequences from our fungal culture collection. Sporulation rates and cumulative spore production of aquatic hyphomycetes showed significant relationships with DIN rather than SRP, with the greatest effects of nutrients found on wood. Cumulative spore production was strongly correlated with plant litter decomposition rates (R2 0.68-0.91). Dominant species of aquatic hyphomycetes were similar as assessed by microscopy and Illumina sequencing. Dissolved nutrients affected fungal community structure based on microscopy (ANOSIM, p=0.001), while the effect of DIN was less pronounced based on Illumina data (PERMANOVA, p=0.02) and not significant for SRP. The effect of substrate (even type of leaf litter when wood was omitted) was greater than the effect of dissolved nutrients in shaping fungal communities. Microscopy appeared to be more sensitive in detecting changes in fungal community structure than NGS. This could be due to (i) microscopy assessing the effects of nutrients on reproductive output of aquatic hyphomycetes while NGS relied on DNA from vegetative fungal biomass embedded in plant litter, and (ii) microscopy focusing on highly active, truly aquatic fungi while NGS also detected DNA from less active, dormant or dead terrestrial fungi initially present on autumn-shed leaves that produced interfering background signals. Due to differences in activity and elemental stoichiometry of fungal species, changes in fungal communities may affect plant litter decomposition and carbon, nutrient, and energy flows in streams.