Coolia is a genus of marine benthic dinoflagellates which is widely distributed in tropical and temperate zones. Toxicity has been reported in selected Coolia species, although the identity of causative compounds is still controversial. In this study, we investigated the taxonomical and toxicological aspects of Coolia species from Brazil. Since light- and electron microscopy-based morphology was not enough to distinguish small-celled species, ITS and LSU D1-D3 phylogenetic analyses were used for species definition. Cultures of Coolia palmyrensis and Coolia santacroce were established from samples collected along the northeastern Brazilian coast, the first record of both species in South Atlantic waters. Cultures of Coolia malayensis and Coolia tropicalis were also established and exhibited acute in vivo toxicity to adults of Artemia salina, while C. palmyrensis and C. santacroce were non-toxic. The presence of 30 yessotoxin analogues, 7 metabolites of Coolia and 44 Gambierdiscus metabolites was screened in 14 strains of Coolia. 44-methyl gambierone (formerly referred to as MTX3) and a new isomer of this compound were detected only in C. tropicalis, using both low- and high-resolution LC-MS/MS. To our knowledge, this is the first report of gambierone analogues in dinoflagellates other than Gambierdiscus; the role of C. tropicalis in ciguatera poisoning thus deserves to be considered in further investigations.