Surface discharges of anthropogenic nutrients historically have been the focus of Florida's water-quality regulations. Groundwater contributions to eutrophication of Florida's surface waters are a more recent focus. Florida's naturally oligotrophic springs, streams, and lakes are experiencing significant anthropogenic nutrient contamination resulting from groundwater discharges with elevated nitrate. Sources of nitrate contamination to these surface-water ecosystems include sewage effluent, industrial animal waste (concentrated animal feedlot operations) and inorganic fertilizers. In this study, stable nitrogen isotope (δ15N) analysis of freshwater macrophytes was combined with basic knowledge of watershed and springshed land use and aquifer characteristics to provide evidence of nitrogen contamination sources and groundwater flowpaths. Selected naturally oligotrophic ecosystems included springs and a spring-run stream within the Ocala National Forest (ONF) and springs, a blackwater stream, and a sinkhole lake on or adjacent to state lands. Elevated δ15N values (∼ + 8 to 12‰) in ONF macrophytes indicated nitrogen contamination from sewage effluent. Underground injections of effluent and other wastes at ONF's Alexander and Juniper Springs Recreation Areas are the sole source of contaminants flowing through the sandy, surficial aquifer at those study areas. Samples from springs on state lands indicated nitrogen contamination from various sources via regional groundwater flowpaths. At Lake Placid's state lands, a dairy-waste lagoon was the groundwater source of nitrogen contamination via the sandy, surficial aquifer. Bulow Creek δ15N macrophyte values (∼ + 5 to 8‰) suggested contamination from both cattle and septic tank leachate. Results indicated that uptake of anthropogenic nitrogen occurred in invasive alien and nuisance native macrophytes in the four freshwater ecosystem types evaluated.