Examining the ecological consequences of the late Quaternary megafaunal extinctions within biodiversity hotspots is crucial for our understanding of the potential consequences of contemporary extinctions. We present the first multi-species record of spores of coprophilous fungi (SCF) from Monquentiva and the high-Andean forests of Colombia to reconstruct Late Pleistocene and Holocene megafaunal abundance. Fossilised pollen and charcoal are used to examine the consequences of megafaunal declines on the surrounding vegetation and fire activity. Our SCF record indicates the presence of Pleistocene megafauna at least since 30,290 BP, with two waves of megafaunal decline at ca. 22,900 BP and 10,990 BP. At Monquentiva, megafaunal decline in the Early Holocene resulted in transitional non-analogue vegetation, loss of some herbivore-dispersed plant taxa, an encroachment of palatable and woody flora, and a rise in fire activity. Differences with other published South-American records suggest that ecological consequences of megafaunal declines were habitat-specific. Overall, we show that ecosystems in the eastern Colombian Cordillera were highly sensitive to the decline of megafaunal populations. Under the current biodiversity crisis, management and conservation efforts must account for the effects of local herbivore declines on plant dispersal, on fire activity, and the potential loss of ecosystem services.