The Spanish conquest of the Cuchumatanes Highlands (1524–1541 ce) led to dramatic land use changes adhering to colonial practices and values, which included the rearing of livestock, agriculture, timber extraction, mining, and the relocation of indigenous populations to new settlements. These changes are often recorded in historical accounts and official records; however, these are sparse, incomplete, and have been lost over the passage of time. Here, we present a high-resolution palaeoenvironmental reconstruction for Cenote Kail (Guatemala) since the Spanish Conquest, to provide additional evidence of land use changes from an integrated multi-proxy perspective. We analysed: (i) fossil pollen; (ii) macroscopic and microscopic charcoal; and (iii) dung fungal spores (Sporormiella) from a lake sediment core extracted from Cenote Kail in the Cuchumatanes highlands, combining these analyses with remotely sensed satellite data. We reconstructed: (i) forest composition and dynamics; (ii) burning, (iii) fauna abundance, and (iv) agricultural activities. High resolution age-depth modelling was conducted using a combination of 210Pb and 14C dates. The high temporal resolution enabled a novel integrated validation of the charcoal data sets with remotely sensed satellite data and the historical record. Three stages of floral compositional change were discerned from the palynological assemblage data encompassing: (i) the decline of mixed hard wood forests (MHWF), associated with the building of new settlements, agriculture and timber extraction for fuel (1550–1675 ce); (ii) pastoral expansions involving the rearing of livestock (1700–1800 ce); and (iii) the expansions of urban settlements and increasing management of the land (1821–2015 ce). Seed predation is suggested as the dominant factor preventing MHWF from re-establishing in the Cuchumatanes Highlands over the past 500 years. Burning is limited locally and regionally and in line with the modern regime, which suggests that fire has been managed and controlled since European contact.