Tropical floodplain lake ecosystems are recognized as important sources of carbon (C) from the water to the atmosphere. They receive large amounts of organic matter and nutrients from the watershed, leading to intense net heterotrophy and carbon dioxide (CO2) emission from open waters. However, the role of extensive stands of floating macrophytes colonizing floodplains areas is still neglected in assessments of net ecosystem exchange of CO2 (NEE). We assessed rates of air-lake CO2 flux using static chambers in both open waters and waters covered by the widespread floating aquatic macrophyte (water hyacinth; Eichornia sp.) in two tropical floodplain lakes in Pantanal, Brazil during different hydrological seasons. In both lakes, areas colonized by floating macrophytes were a net CO2 sink during all seasons. In contrast, open waters emitted CO2, with higher emissions during the rising and high water periods. Our results indicate that the lake NEE can be substantially overestimated (fivefold or more in the studied lakes) if the carbon fixation by macrophytes is not considered. The contribution of these plants can lead to neutral or negative NEE (that is, net uptake of CO2) on a yearly basis. This highlights the importance of floating aquatic macrophytes for the C balance in shallow lakes and extensive floodplain areas.