The Juréia-Itatins Ecological Station (JIES) is one of the few protected areas in the state of São Paulo, which harbor a significant portion of the Atlantic forest. Although there is high diversity and endemism of fish species in this region, knowledge of the ichthyofauna in JIES is incipient. A total of 4,809 specimens belonging to six orders, 15 families and 31 species were captured during April 2009 to February 2010, using electrofishing, gillnets and traps. The family Characidae was predominant, mainly due to the abundance of freshwater species such as Astyanax ribeirae, Mimagoniates microlepis, and Hollandichthys multifasciatus. Although the estuarine regions were rarely sampled in this study, six brackish water species were captured. In this study, nine species previously unknown from JIES were discovered. Five endangered fish species, endemic to the Atlantic forest, occur at JIES, thus the presence of protected areas, as a conservation measure, is particularly relevant.