The early Eocene Climatic Optimum (EECO; ∼53–49 Ma) was the warmest sustained global warming episode of the Cenozoic, accompanied by major alterations in terrestrial and marine biota. Here we detail changes in low-latitude calcareous nannofossil assemblages at two tropical sites in the equatorial Atlantic (ODP Site 1258) and Pacific (ODP Site 865), across the EECO and the subsequent early to middle Eocene cooling transition (EMET, ∼49–47 Ma). Our results show that the evolutionary turnover between Toweius and Reticulofenestra is characterized by a two-step decline in Toweius marking peak warmth during the EECO (52–50 Ma). Tropical nannofossil assemblages reveal a broad acme of Discoaster during the same time, as previously reported at several sites, but also uniquely show distinct abundance increases in specialist taxa such as Campylosphaera, Umbilicosphaera, and Calcidiscus. The decline of these taxa and the rapid expansion of Reticulofenestra characterized the conclusion of the EECO and the start of global temperature decline ∼49 Ma. Multivariate statistical analysis of these and nine other nannofossil data sets from higher latitudes reveals that the occurrence and prevalence of specialist taxa constitutes a tropical signature not reported elsewhere, in addition to the previously identified latitudinal expansion of (sub)tropical taxa during the EECO. This pronounced compositional contrast was likely enabled by enhanced thermal stratification, sluggish ocean circulation, and intensified hydrological dynamics of the tropics during the EECO. Global cooling and more vigorous meridional overturning circulation after the EECO likely explain increased connectivity, biogeographical restructuring, and the establishment of the first Reticulofenestra-dominated coccolithophore communities.