Endophytic fungi live in the interior of healthy plants without causing them any damage. These fungi are of biotechnological interest; they may be used in the biological control of pests and plant diseases, and in the pharmaceutical industry. The aquatic macrophytes Eichhornia azurea (Kunth) and Eichhornia crassipes (Mart.) belong to the Pontederiaceae family. The first is a fixed-floating species and the second is a free-floating species that is known for its phytoremediation potential. The fungal endophytes associated with the leaves of E. azurea and E. crassipes, native to the Upper Paraná River floodplain, Brazil, were isolated. The sequencing of the ITS1-5.8S-ITS2 region of ribosomal DNA was performed and the nucleotide sequences obtained were compared with those available in the GenBank database for the molecular identification of the isolates. The construction of phylogenetic trees was performed using the MEGA5 software. The results showed that high colonization frequencies were obtained from the 610 foliar fragments sampled from each plant: 87.86% for E. azurea and 88.85% for E. crassipes. At the genus level, it was possible to identify 19 fungal endophytes belonging to the genera Alternaria, Bipolaris, Cercospora, Diaporthe, Gibberella, Pestalotiopsis, Plectosphaerella, Phoma, and Saccharicola. Two other endophytes were identified at the species level (Microsphaeropsis arundinis). Genera Bipolaris, Cercospora, Microsphaeropsis, and Phoma were found as endophytes in the two macrophytes and the other genera were host-specific, being isolated from only one macrophyte, proving that there is a small difference in the endophytic diversity of the two Eichhornia species analyzed.