Empirical monitoring studies of catchment-scale Escherichia coli burden to land from agriculture are scarce. This is not surprising given the complexity associated with the temporal and spatial heterogeneity in the excretion of livestock faecal deposits and variability in microbial content of faeces. However, such information is needed to appreciate better how land management and landscape features impact on water quality draining agricultural landscapes. The aim of this study was to develop and test a field-based protocol for determining the burden of E. coli in a small headwater catchment in the UK. Predictions of E. coli burden using an empirical model based on previous best estimates of excretion and shedding rates were also evaluated against observed data. The results indicated that an empirical model utilising key parameters was able to satisfactorily predict E. coli burden on pasture most of the time, with 89% of observed values falling within the minimum and maximum range of predicted values. In particular, the overall temporal pattern of E. coli burden on pasture is captured by the model. The observed and predicted values recorded a disagreement of > 1 order of magnitude on only one of the nine sampling dates throughout an annual period. While a first approximation of E. coli burden to land, this field-based protocol represents one of the first comprehensive approaches for providing a real estimate of a dynamic microbial reservoir at the headwater catchment scale and highlights the utility of a simple dynamic empirical model for a more economical prediction of catchment-scale E. coli burden.