Coeur d’Alene Lake (the Lake) has received significant contamination from legacy mining. Aquatic macrophytes provide important ecosystem services, such as food or habitat, but also have the ability to accumulate contaminants. We examined contaminants (arsenic, cadmium, copper, lead, and zinc) and other analytes (e.g., iron, phosphorus, and total Kjeldahl nitrogen (TKN)) in macrophytes from the Lake. Macrophytes were collected in the Lake from the uncontaminated southern end to the outlet of the Coeur d’Alene River (main contaminant source) located northward and mid lake. Most analytes showed significant north to south trends (Kendall’s tau p ≤ 0.015). Concentrations of cadmium (18.2 ± 12.1), copper (13.0 ± 6.6), lead (195 ± 193), and zinc (1128 ± 523) were highest in macrophytes near the Coeur d’Alene River outlet (mean ± standard deviation in mg/kg dry biomass). Conversely, aluminum, iron, phosphorus, and TKN were highest in macrophytes from the south, potentially related to the Lake’s trophic gradient. Generalized additive modelling confirmed latitudinal trends, but revealed that longitude and depth were also important predictors of analyte concentration (40–95% deviance explained for contaminants). We used sediment and soil screening benchmarks to calculate toxicity quotients. Quotients were used to assess potential toxicity to macrophyte associated biota and delineate where macrophyte concentrations exceeded local background concentrations. Exceedances (toxicity quotient > one) of background levels by macrophyte concentrations were highest for zinc (86%), followed by cadmium (84%), lead (23%), and arsenic (5%).