Increasing nitrate concentrations in many large karstic spring systems throughout northern Florida have raised both ecosystem and human health concerns.  To better understand sources of nitrate in two basins, multiple chemical constituents (N, O, H, C isotopes; 64 organic wastewater compounds (OWCs); 16 pharmaceutical compounds; nutrients), and microbiological indicators were used to assess the impact of the land application of wastewater on water quality in the Upper Floridan aquifer.  The 960 km² Ichetucknee springs basin (ISB) contains a 142-ha sprayfield site where 6,800 cubic meters per day (m³/day) of treated sewage effluent are applied.  The 2,980 km² Wakulla springs basin (WSB) contains an 810-ha sprayfield site where 76,000 m³/day of treated municipal wastewater are applied. Water samples were collected from the effluent reservoir, monitoring wells, and springs in the ISB during low- and high-flow conditions in 2005, and in the WSB during base-flow conditions in 2005 and 2006. Lower delta ¹⁸O and ²H values, and lower boron, and chloride concentrations in water from monitoring wells relative to effluent reservoir water at both sites indicated more than a 50 percent dilution due to mixing with ambient ground water.  Tracer concentrations decreased substantially in ground-water and spring-water samples with distance downgradient from both sprayfields and indicated 10- to 15-fold dilution factors relative to reservoir concentrations.  Concentrations of OWCs in effluent reservoir waters were near method reporting limits [low micrograms-per-liter (ug/L)].  The insect repellent, N,N-diethyl-metatoluamide (DEET) was detected at most sampled sites (<0.5 to 5.4 ug/L). Elevated levels of total coliforms and enterococci were found in ground water at two ISB sites during high-flow conditions.  Detections of OWCs and pharmaceutical compounds (typically <0.5 ug/L) and elevated nitrate concentrations (>1mg/L) in ground water at sites throughout each basin indicate that the Upper Floridan aquifer is highly vulnerable to contamination from several anthropogenic sources.