Macrophytes are fundamental photosynthetic organisms for functioning freshwater ecosystems, identified as potential bioindicators of mercury (Hg) in the environment. We quantified the concentrations of total Hg (THg) in water and macrophytes (Paspalum repens Berg.) from 17 lakes on the Araguaia River floodplain, aiming to compare the bioconcentration factor (BCF) in the aerial tissues and roots; evaluate the translocation factor (TF) between plant tissues; and assess the influence of environmental factors and land use on THg concentrations in water and macrophytes. The BCF was significantly higher in roots (1.29 ± 0.32) than in aerial tissues (0.41 ± 0.34), with low TF between plant tissues (0.14 ± 0.06). The highest concentrations of THg in water were determined in lakes with higher land use intensity and a pH close to neutral, indicating the transport of particulate-bound Hg and the immobilization in the water column. In contrast, wetlands were priority areas for the bioconcentration of THg in macrophytes, associated with sulfate, dissolved oxygen, and oxidation–reduction potential in the water. Thus, although P. repens is not a suitable bioindicator of Hg mobilization by anthropogenic land use in our study area, our results suggest the potential of macrophytes as bioindicators of sites that are favorable to Hg methylation.