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Paleolimnological evidence indicates the ancient Maya transformed terrestrial ecosystems by felling forest vegetation to construct large civic-ceremonial centers and to expand agriculture. Human settlements influenced lacustrine environments but the effects of Maya activities on aquatic ecosystems remain poorly studied. Here we analyzed a sediment core from Lake Petén Itzá, Guatemala, to infer paleoenvironmental changes resulting from Maya occupation of the archaeological site of Nixtun-Ch’ich’. Increases in charcoal and fecal stanol concentrations indicate Maya occupation of the Candelaria Peninsula by the late Early Preclassic period. Geochemical proxies reveal a period of lake ecosystem alteration during construction and expansion of the city’s urban grid in the Middle and Late Preclassic periods. Depopulation of the city in the Terminal Preclassic resulted in a decline in lake trophic state. Whereas previous studies of Petén waterbodies have indicated depressed lacustrine primary production, the core collected near Nixtun-Ch’ich’ shows evidence of ancient Maya lake ecosystem deterioration.