Fungi are essential in aquatic ecosystems, transforming organic matter into energy sources that support higher trophic levels. However, researchers do not yet know the extent of fungal diversity and species distribution within these important ecosystems. Therefore, we examined the detrital fungal communities from contrasting aquatic habitats (temperate peatlands and streams) to provide an in-depth inventory and greater understanding of how these communities differ. Fine submerged detritus or substrate on or beneath the stream bed were collected from 6 sites. Fungal cultures were isolated from samples collected in May, July/August, and November from 2 sites in 2014 and 4 sites in 2016. Culture-independent analyses were conducted on 42 environmental samples collected in November 2016. Results indicated that peatland and stream fungal communities were taxonomically diverse, phylogenetically distinct, and harbored many unknown taxa from the kingdom Fungi. Specifically, stream habitats were more species-rich, in both number of species and phylogenetic diversity, compared to peatland habitats. In addition, fungal species and phylogenetic distribution within most major fungal classes were distinct between peatland and stream fungal communities. In light of global climate change, habitat loss, and water pollution, it has become increasingly important to examine these understudied and essential fungal communities within these ecosystems.