As the requirements of the Water Framework Directive (WFD) and the US Clean Water Act (USCWA) for the maintenance of microbiological water quality in ‘protected areas’ highlight, there is a growing recognition that integrated management of point and diffuse sources of microbial pollution is essential. New information on catchment microbial dynamics and, in particular, the sources of faecal indicator bacteria found in bathing and shellfish harvesting waters is a pre-requisite for the design of any ‘programme of measures’ at the drainage basin scale to secure and maintain compliance with existing and new health-based microbiological standards. This paper reports on a catchment-scale microbial source tracking (MST) study in the Leven Estuary drainage basin, northwest England, an area for which quantitative faecal indicator source apportionment empirical data and land use information were also collected. Since previous MST studies have been based on laboratory trials using ‘manufactured’ samples or analyses of spot environmental samples without the contextual microbial flux data (under high and low flow conditions) and source information, such background data are needed to evaluate the utility of MST in USCWA total maximum daily load (TMDL) assessments or WFD ‘Programmes of Measures’. Thus, the operational utility of MST remains in some doubt. The results of this investigation, using genotyping of Bacteroidetes using polymerase chain reaction (PCR) and male-specific ribonucleic acid coliphage (F + RNA coliphage) using hybridisation, suggest some discrimination is possible between livestock- and human-derived faecal indicator concentrations but, in inter-grade areas, the degree to which the tracer picture reflected the land use pattern and probable faecal indicator loading were less distinct. Interestingly, the MST data was more reliable on high flow samples when much of the faecal indicator flux from catchment systems occurs. Whilst a useful supplementary tool, the MST information did not provide quantitative source apportionment for the study catchment. Thus, it could not replace detailed empirical measurement of microbial flux at key catchment outlets to underpin faecal indicator source apportionment. Therefore, the MST techniques reported herein currently may not meet the standards required to be a useful forensic tool, although continued development of the methods and further catchment scale studies could increase confidence in such methods for future application.