The Bolivian Chiquitano dry forest is the largest block of intact seasonally dry tropical forest in South America and is a priority ecoregion for conservation due to its high threat status. However, the long-term impacts of drier climatic conditions on tropical dry forests are not well understood, despite climate models predicting increased droughts over Bolivia in the coming century. In this paper, we assess the impacts of drier climatic conditions during the mid-Holocene on the Bolivian Chiquitano tropical dry forest using fossilised pollen, phytoliths, macro-charcoal, and geochemical proxies from a sediment core from a large lake (Laguna Mandioré) on the Bolivia–Brazil border. Our results show that drier climatic conditions during the mid-Holocene caused a local-scale, ecotonal expansion of upland savannah at the expense of dry forest. Interaction between drier climatic conditions and fire regime likely exerted a stronger control over the position of the dry forest–savannah ecotone than edaphic factors. However, the majority of the dry forest within the lake catchment maintained a closed canopy throughout the drier conditions of the mid-Holocene, despite floristic turnover towards more drought-tolerant taxa. These findings imply overall resilience of the Chiquitano dry forest biome to future drought, albeit with floristic changes and upland savannah encroachment at ecotones.