The Tirimbina Biological Reserve (TBR), located in La Virgen de Sarapiquí, Heredia Province, Atlantic versant (Sarapiquí river basin), Costa Rica, is a private wildlife refuge established as “a place of absolute conservation” and forscientific research. An annotated and illustrated catalog of the fish species known to occur within the limits of the TBR, as well as an identification key to and descriptions of all species listed, is presented. A total of 29 species, 23 genera, 9 families and 6 orders were recorded from 13 localities regularly sampled between December 2009 and December 2016. The conservation status of the species and major threats also are briefly discussed. This contribution will serve as a scientific and educational tool to researchers and conservationists, as well as to the public, interested in knowing and working with the fish fauna of the TBR and surrounding areas.