We present the first palaeolimnological investigation of chironomid larval assemblages from the Bolivian Eastern Cordillera. Taxonomic diagnoses are provided for the 10 chironomid taxa (subfamilies: Chironominae, Orthocladiinae and Tanypodinae) identified in the lake sediments. We compared changes in the chironomid assemblage from two Andean sites with previously reported palynological, charcoal and geochemical data, and highlight the potential of chironomid analysis to provide additional insights into environmental change in this region of high biodiversity over the last 18,000 years. At Lake Challacaba (17°33.257′S, 65°34.024′W; 3,400 m asl), the chironomid and geochemical data indicate periodic desiccation and hypersalinty of the basin c. 4,000–3,460 cal year BP. Increased abundance of Chironomus sp. at c. 1,000 cal year BP suggests a change in human activity, supporting inferences from the pollen and spore records, which indicate elevated pastoral agriculture at this time. The greatest assemblage change in the chironomid record from Laguna Khomer Kocha Upper (17°16.514′S, 65°43.945′W; 4,153 m asl) occurred at c. 6,380 cal year BP, concomitant with an increase in marsh woodland taxa, wetter conditions and a rising lake level at the end of a Holocene dry event. There is no apparent response in the chironomid assemblage to burning, however, at the onset of this dry event (c. 10,000 cal year BP), which is the major transformative agent of the terrestrial vegetation. This study shows that chironomid assemblages in the tropical Andes responded to regional and local environmental changes, and in particular, that they were sensitive to adjustments in net moisture balance (water level fluctuations and salinity) and anthropogenic impacts (nutrient input). This suggests that within-lake processes are more important as drivers of chironomid assemblage composition than terrestrial vegetation or fire regime. Nevertheless, the full potential of subfossil chironomid analysis will only be realised once more modern autecological data are available.