In a world of increasingly urbanized environments, it is critical to understand the impact on microbial communities, including fungal communities, as a measure of ecosystem health, and to document how these environments are changing. Aquatic environments, in particular, can be highly sensitive to urbanization with removal of local habitat and inputs of wastewater and contaminants, leading to a displacement or extinction of natural flora, fauna, and funga. Aquatic microbial communities, especially fungal communities, are extremely complex and far less characterized compared to their macro counterparts. Here, we characterized the fungal microbiome, i.e. \"mycobiome\", across multiple habitats (water, sediment, and amphibian fecal matter) from three species (American bullfrog, Green frog and Pickerel frog) within the Tatnuck Brook waterway in Worcester, MA. The Tatnuck Brook waterway is a connected group of streams, ponds, and lakes that transition from protected areas at its headwaters, below the city\'s drinking water reservoir, to highly urbanized regions within Worcester. Using metabarcode sequencing, we found that water, sediment, and host habitats harbor distinct fungal communities, but all exhibit parallel shifts in diversity along an urbanization gradient. Furthermore, we identified fungal taxa from the amphibian mycobiome and environmental samples that exhibited a range of sensitivities to urbanization. These taxa, including Basidiobolus, Cladosporium, and Lemonniera in fecal and sediment samples, as well as Candida, which was found across all habitats, are potential indicators of shifts in aquatic ecosystems due to urbanization. Their responses to urban stressors may serve as a baseline for further studies of aquatic fungal communities.