Abstract The Paranaguá Estuarine Complex (PEC) is one of Brazil's largest southwest Atlantic estuarine systems, possessing a rich microalgae diversity that remains to be fully explored. Therefore, due to the increasing interest in the microalgae biotechnological potential, this study isolated and identified diatoms found in the PEC. The diatoms were purified and analyzed with light and scanning electron microscopy for morphological identification, while DNA sequences were used for molecular identification. Although a diatoms rich diversity was obtained, only a few were viable after the cultivation period. The two best-selected strains were identified as belonging to two genera, Nitzschia and Navicula. The rbcL region was found to be the most informative for species identification. Morphological and molecular analyses allowed for the identification of species Nitzschia inconspicua and Navicula pseudoantonii, which was understood as the first report of N. pseudoantonii in the American continent.