Aquatic fungi are important decomposers of plant litter in temperate streams. However, these microorganisms have been poorly studied in tropical streams. We assessed the dynamics of aquatic hyphomycetes during the decomposition of Ficus sp. (Moraceae) leaves in an intermittent lowland stream of northwestern Venezuela. Our goals were to: 1) determine the composition of aquatic hyphomycetes during leaf decomposition, 2) assess the effect of different mesh sizes (i.e., coarse vs fine mesh) on assemblage composition and activity, and 3) assess the role of habitat (i.e., pools vs riffles) on fungal assemblages. Coarse- and fine-mesh litter bags were incubated in a pool and a riffle for 31 d. For each habitat and treatment, we determined physicochemical variables, sporulation activity, and the structure and composition of fungal assemblages. Leaf litter decomposition rates ranged from 0.058/d in fine-mesh bags in the riffle to 0.031/d in coarse-mesh bags in the pool. Decomposition rates were significantly influenced by habitat type, with the highest decomposition rates found in riffle habitats. Sporulation rates were significantly different between habitats but not between mesh sizes. The highest sporulation rate (399 spores mg⁻¹ leaf dry mass d⁻¹) and fungal diversity (16 spp.) was found in coarse-mesh bags in the riffle. Twenty-one fungal species were identified during the study. Anguillospora longissima and Lunuluspora curvula contributed up to 75% of the conidia produced in the coarse- and fine-mesh bags in the riffle, whereas Filosporella aquatica and Lemonniera terrestris accounted for 65 to 75% of the conidia produced in the coarse- and fine-mesh bags in the pool. In ordination space based on species composition, fungal assemblages formed 2 clear groups that corresponded to habitat type. A temporal change in fungal species composition was observed in riffle samples. Early succession assemblages (2–14 d) were characterized by the presence of A. longissima, L. curvula, and F. aquatica, whereas late assemblages (24–31 d) were dominated by Pyramidospora fluminea, Pyramidospora casuarinae, and unidentified sp 4. Overall, habitat type (pool or riffle) influenced sporulation rates and assemblage composition of aquatic hyphomycetes. Our study suggests that environmental factors associated with hydrology (e.g., stream velocity, width, depth) regulate fungal composition and activity and, hence, leaf litter decomposition in this tropical stream.