The Amazon basin is a major component of the global carbon and hydrological cycles, a significant natural source of methane, and home to remarkable biodiversity and endemism. Reconstructing past climate changes in the Amazon basin is important for a better understanding of the effect of such changes on these critical functions of the basin. Using a novel biomarker proxy, based on the membrane lipids of soil bacteria with a new regional calibration, we present a reconstruction of changes in mean annual air temperatures for the Amazon catchment during the last 37 kyr B.P. Biomarkers were extracted from Ocean Drilling Program sediment core ODP942 recovered from the Amazon fan. The Amazon fan is a major depository for terrestrial sediments, with the advantage that the terrestrial material captured reflects a regional integration of the whole river catchment. The reconstructed tropical Amazonian temperatures were ∼5°C cooler at the Last Glacial Maximum (∼21°C) compared to modern values (∼26°C). This is in agreement with previous estimates of tropical continental temperatures in the tropical Amazon basin and tropical Africa during the Last Glacial Maximum. Moreover, we also illustrate how the soil bacterial membrane lipid record reveals major changes in basin dynamics and sediment provenance during the glacial-Holocene transition, impacting the biomarker reconstructions from ∼11 kyr onward.