This study focused on freshwater hyphomycetes associated with submerged decaying leaves of Calophyllum brasiliense in three streams in the semiarid region of Brazil. Leaves were enclosed in litter bags and deployed into streams on two occasions (dry and wet seasons). Samples of leaf litter were collected every two months (November 2013 to January 2015). Unlike other studies, we specifically targeted ecologically distinct groups of freshwater hyphomycetes by using two methods to induce fungal sporulation: submerged incubation (SI) of leaf litter on an orbital shaker and incubation in moist chambers (MC). We aimed to analyze and compare the structure of freshwater hyphomycete communities in the streams using both methods, detect possible successional patterns, and evaluate if environmental variables have influenced fungal diversity and sporulation rates. Sixty-nine taxa of freshwater hyphomycetes were observed. Of these, 26 were found under SI and 56 in MC. We observed large differences in fungal communities recovered by SI vs. MC that demonstrates the importance of using several methodological approaches to maximize the number of taxa recovered in ecological studies of litter-associated fungi. The highest sporulation rates under SI were observed during the wet season. Results of community ordination suggested that environmental variables affected the structure of fungal communities; for example, water velocity showed a positive effect on fungal diversity, while higher oxygen availability was associated with lower diversity. This study advances our understanding of the freshwater hyphomycete communities in tropical streams where ecological studies of aquatic fungi are still uncommon.