Infections caused by waterborne Escherichia coli O157 continue to pose a public health risk. An increase in faecal coliform loading of watercourses due to expanding populations, intensification of agriculture and climate change are predicted to amplify these risks. Understanding the effect of land use on the ecology of E. coli O157 in environmental waters is therefore important for implementing effective mitigation measures. In order to test the hypothesis that activity of waterborne E. coli O157 is affected by both land use type and the respective autochthonous microbial communities, we inoculated replicate microcosms of water collected from areas of contrasting land uses within a catchment with a chromosomally lux-marked E. coli O157. Pathogen metabolic activity and its ability to reactivate following addition of nutrients were quantified over time in both filter-sterilised and non-sterile microcosms. Metabolic activity differed significantly according to the land use type, the degree of competition from background microbes and the availability of nutrients. These results indicate that land use types associated with particular areas of a watercourse should be considered a central factor in models that aim to predict pathogen risk in environmental waters.