Aims: Microbial water quality and possible human sources of faecal pollution were assessed in a Florida estuary that serves shellfishing and recreational activities. Methods and Results: Indicator organisms (IO), including faecal coliforms, Escherichia coli and enterococci, were quantified from marine and river waters, sediments and oysters. Florida recreational water standards were infrequently exceeded (6-10% of samples); however, shellfishing standards were more frequently exceeded (28%). IO concentrations in oysters and overlaying waters were significantly correlated, but oyster and sediment IO concentrations were uncorrelated. The human-associated esp gene of Enterococcus faecium was detected in marine and fresh waters at sites with suspected human sewage contamination. Lagrangian drifters, used to determine the pathways of bacterial transport and deposition, suggested that sediment deposition from the Ochlockonee River contributes to frequent detection of esp at a Gulf of Mexico beach. Conclusions: These data indicate that human faecal pollution affects water quality in Wakulla County and that local topography and hydrology play a role in bacterial transport and deposition. Significance and Impact of the Study: A combination of IO enumeration, microbial source tracking methods and regional hydrological study can reliably inform regulatory agencies of IO sources, improving risk assessment and pollution mitigation in impaired waters.