The Gurgueia River in southern Piauí state, Brazil, is the largest affluent on the right margin of the Parnaíba River basin. This study aimed to inventory the Gurgueia River ichthyofauna, and contribute to the knowledge of fish diversity in Northeastern Brazil. We sampled 71 locations throughout the Gurgueia sub-basin between 2006 and 2014, which resulted in 90 fish species representing 67 genera, 26 families and six orders, including two non-native species. Six species are newly recorded for the Parnaíba basin (Hasemania nana, Hemigrammus brevis, H. guyanensis, H. ora, Corydoras sp. and Cetopsorhamdia sp.), increasing its freshwater ichthyofauna to 152 species. The Gurgueia River contains 59.2% of the ichthyofauna known for the Parnaíba basin, and can be used as a reference point for the establishment of priority areas for the conservation of the freshwater fish fauna of the Brazilian Northeast.