A species richness hotspot of ant-mimicking (or myrmecomorphic) spiders of the subfamily Castianeirinae Reiskind, 1969, is reported from the premontane Chiquitano forest at the Bolivian orocline. In a transect of 350 m in a forest fragment of ~10 ha, 148 individuals of five genera and 10 species were collected, including the first country records for the genera Mazax O. Pickard-Cambridge, 1898, Myrmecotypus O. Pickard-Cambridge, 1894, and Sphecotypus O. Pickard-Cambridge, 1895. The observations from this study represent an increase of the known fauna of Bolivian Castianeirinae from two to five genera and from three to at least 12 species. The generic and species richness is comparable with the fauna of mega-diverse moist forests in other Neotropical countries and may result from overlapping distributional ranges of faunal elements from several ecoregions. The discovery of this hotspot in an easily accessible and small area provides an excellent opportunity to gain further insight into the factors determining the evolutionary ecology and ant mimicry in Castianeirinae communities.