This study investigated the sources of stormwater fecal contamination in Antelope Creek watershed (Lincoln, NE). Stormwater samples from eight different storm drain outfalls in three events and potential source samples (street sweepings, erodible watershed soil, sanitary sewage, and feces of pigeon, swallow, duck, small mammals, dog, and horse) were collected for analysis. The V4 region of the 16S rRNA gene in the DNA extracted from each sample was sequenced and analyzed to identify microbial communities. SourceTracker analysis, carried out based on microbial community, attributed street sweepings as the largest contributor of the fecal indicator population (Bacteroidetes phylum) in stormwater. DNA extracted from the water samples and sanitary sewage were analyzed using the HF183/BacR287 qPCR assay to quantify human fecal contamination. Both SourceTracker analysis and HF183/BacR287 assay revealed sanitary sewage as a minor contributor of contamination in stormwater suggesting there may be sewage exfiltration and/or cross connection in the study area.