A checklist of the marine and estuarine fishes of the inner part of the Gulf of Nicoya, Pacific coast of Costa Rica, Central America, was compiled by examining a museum fish collection, resulting in 72 families and 274 species. Of these species, 127 (46.4%) were marine species and 147 (53.6%) were estuarine-associated species. In terms of their life history and considering the habitat type classification, 188 (almost 70% of the total) were categorized as species inhabiting soft-bottom habitats, reflecting the large estuarine environment and rich fish diversity of the Gulf despite its relatively small area in the tropical Eastern Pacific region. Furthermore, the list contains 13 threatened species of IUCN Red List, which need further research to understand their abundance and their exposure to habitat loss in the Gulf. Further detailed studies on its fish fauna and habitat are needed to better understand and conserve biodiversity within the whole Gulf.