The large number of less-known coastal drainages in the Atlantic Forest still represents an important gap in the knowledge of ichthyofauna. It is evaluated the taxonomic diversity, frequency and constancy of occurrence, and the distribution of species in the various segments of the Perequê-Açu river basin, state of Rio de Janeiro, Brazil. Twelve sampling campaigns resulted in 13,423 individuals, belonging to seven orders, 10 families, 21 genera, and 23 native species. Cyprinodontiformes and Siluriformes represented about 87.2% of the total abundance, mainly due to the contribution of Phalloceros anisophallos and Schizolecis guntheri, which account for 63.2% of the total sampled individuals. The analysis of constancy of occurrence reveals that 78.3% of the drainage species are residents, with approximately 93% of the residents of the Carrasquinho River sub-basin. The Sertões River sub-basin had the largest number of species in total: 18. The rarefaction curve did not reach an asymptote, although Chao 2 and bootstrap estimate the richness of 23.9 species and 23.7 species, respectively. The results of PERMANOVA, PCoA, and SIMPER indicate that the fish composition along the catchment is not homogeneous. The conservation status of species is also discussed, regionally, nationally, and globally.