Bayou Lafourche, which is the sole drinking water source for 300,000 people in Louisiana, has failed to consistently meet its designated use criteria set by the Louisiana Department of Environmental Quality (LDEQ). This resulted in a total maximum daily load to be imposed on the Bayou by the Environmental Protection Agency (EPA). These designated use water quality criteria include fecal coliform (FC) levels for drinking water source, primary contact recreation, and secondary contact recreation. The goal of this study was to identify and enumerate anthropogenic nonpoint source FC contamination from malfunctioning home sewage systems in the Bayou's watershed. Thirty-four sites along the Bayou were selected for the study. Samples were analyzed for optical brightener ratios, FC CFU/100 mL (mFC), Escherichia coli, and three human markers, including human polyomavirus BK, the Archaeon Methanobrevibacter smithii, and the human-associated Brevibacterioides HF 183 eubacteria. Frequencies of sites with all three anthropogenic molecular markers are considered positive for human fecal contamination. This study provided data to address the problem of malfunctioning on-site sewage systems in the Bayou Lafourche watershed.