Among nitrogen species, nitrate is more stable than ammonium and nitrite, and it is an important nitrogenous pollutant in surface water. However, little is known about the characterization of epiphytic microbial communities on submersed macrophytes under nitrate loading. In this study, we investigated the co-occurring pattern and response of bacteria and microeukaryotes in epiphytic biofilms under nitrate loading. Nitrate loading significantly affected bacterial and eukaryotic communities, and turnover played greater contribution to the total dissimilarity than nestedness by partitioning beta-diversity analysis. Cyanobacteria, α-proteobacteria, β-proteobacteria, Actinobacteria, Planctomycetes, Bacteroidetes, and γ-proteobacteria were dominant bacterial phyla/classes. Metazoan (phylum Arthropoda, Rotifera, Gastrotricha, Annelida, and Nematoda) and algae (phylum Bacillariophyta, Chlorophyta, and Streptophyta) were dominated in eukaryotic communities. The abundances of denitrifying bacteria (Rhodobacter, Acinetobacter, Bacillus, Flavobacterium, and Pseudomonas) and genes (nirS, cnorB, and nosZ) increased with nitrate loading. The network analysis showed there were complex interactions among photosynthetic microbes, metazoan, and bacteria (including denitrifiers) that they were potentially interrelated via photosynthesis, predation or feeding. This study provides new perspectives into understanding the factors affecting nitrate removal mechanisms in wetlands with submersed macrophytes.