The coastal wetland ecosystems in Florida are highly sensitive to changes in freshwater budget, which is driven by regional sea surface temperature, tropical storm activity, and the El Niño–Southern Oscillation (ENSO). Although studying Florida wetlands is pivotal to the understanding of these interacting climate systems, wetland dynamics have been severely altered by recent land use and drainage activities. To gather insights into the natural variability of the coastal ecosystems in Florida versus the effects of anthropogenic impact in the area, we present a 300-year record of changes in the hydrological cycle from a shallow subtropical estuary (Rookery Bay) on the western shelf of Florida, Gulf of Mexico. Palynological (pollen and organic-walled dinoflagellate cysts), diatom, and sedimentological analyses of a sediment core reveal significant changes in past runoff and wetland development. The onset and development of human impact in Florida are evident from high influx of Ambrosia pollen at about A.D. 1900, indicative of land clearance and disturbed conditions. To date, this is the southernmost record of Ambrosia increase related to human impact in the United States. Wetland drainage and deforestation since A.D. 1900 are evident from the reduced freshwater wetland and pine vegetation, and lower abundances of phytoplankton species indicative of lagoonal and brackish conditions. High runoff after A.D. 1900 relates to increased erosion and may correspondingly reflect higher impact from hurricane landfalls in SW Florida. Several phases with high siliciclastic input and greater wetland pollen abundance occur that predate the human impact period. These phases are interpreted as periods with higher runoff and are likely related to regional longer-term climate variability.