AIM: Identify relationships among microbial variables [fecal indicator bacteria (FIB) and microbial source tracking (MST) marker genes] and nutrients to improve source identification in two polluted, freshwater streams in Florida. METHODS AND RESULTS: Water and sediment were sampled at Bullfrog Creek (BFC) and Sweetwater Creek (SWC), which varied in land use and expected sources of fecal pollution. Escherichia coli and enterococci were cultured, sewage-associated HF183 and bird-associated GFD genetic markers were assessed by quantitative polymerase chain reaction (qPCR), and organic and inorganic nutrients were analyzed. Escherichia coli and enterococci exceeded recreational water quality criteria in 47% and 85% of samples, respectively, at less urbanized BFC, and in 8% and 62% of samples at more urbanized SWC. HF183, but not GFD, was positively associated with surface water nitrate by multivariate analysis and binary logistic regression. Sediment organic matter was lowest at urbanized sites in both streams and inversely associated with surface water FIB. CONCLUSIONS: Measuring nutrients alongside FIB and MST revealed that nitrate levels in water, but not phosphorus or organic carbon levels, were predictive of sewage pollution.