ABSTRACT We analyzed structural attributes (epiphyton composition, richness, density, biomass, and life forms) involved in interactions between the epiphyton and Characeae (Chara and Nitella) in 19 tropical reservoir environments, 7 with the presence of Chara, 7 with Nitella, and 5 with both genera. A sampling station measuring 4 m² was defined in each environment for the collection of abiotic and biotic data without temporal repetition. Univariate and multivariate analyses were performed for the characterization of each environment. Regression analysis and partial redundancy analysis (pRDA) were used to test the substrate–epiphyton relationship. The substrate biomass was significantly associated (p < 0.05) with epiphyton biomass, total density, and filamentous forms. The pRDA revealed that epiphyton biomass was related to the 2 substrates, whereas composition was related to the environment (p < 0.05). A negative association was found between Chara and Spirogyra (36.2%). Nitella was positively associated with the genera Lyngbya, Oedocladium, and Oedonogium and negatively with Staurastrum, whose genera together accounted for 12.9% of the biomass of the community. Characeae (Chara and Nitella) did not exert a direct influence on total richness or composition; these attributes were explained by environmental factors. Chara and Nitella biomass had a positive interaction with epiphyton biomass and density, but each substrate promoted specificity and distinct intraspecific relationships in the epiphytic structure in the tropical reservoir environments.