The dataset contains Esherichia coli and quantitative polymerase chain reaction data for microbial source tracking markers in the Chattahoochee River and streams within the Chattahoochee River National Recreation area from samples collected in the winter and summer of the federal fiscal years 2016 and 2017. Six water samples were collected from three sites on the Chattahoochee River each season. Eleven stream sites and one additional site on the Chattahoochee River were sampled once each season, with the exception of two sites, which were not sampled in winter FY16.