Diffuse microbial pollution from agriculture is a key contributor to water quality impairment.Reducing the risk of microbial contamination of watercourses from agricultural sources requires both environmentally appropriate and socially acceptable mitigation and management approaches. A cross-disciplinary toolkit for on-farm microbial risk assessment is presented that can represent both social and environmental factors promoting or preventing the accumulation of faecal indicator organisms (FIOs) within the farm environment, and also their subsequent transfer to watercourses. Four key risk criteria were identified as governing FIO loss from land to water. These were 'accumulating E. coli burden to land', 'landscape transfer potential', 'infrastructure' and 'social and economical obstacles to taking action'. The toolkit can be used to determine (i) the relative risk of a farm enterprise contributing to microbial watercourse pollution and (ii) appropriate and targeted mitigation to reduce the risk of FIO loss from land to water. A comparison of the toolkit output with microbiological water quality draining from three contrasting grassland farm enterprises provided a preliminary evaluation of the prototype approach. When applied to 31 grassland farm enterprises the toolkit suggested that 0% were categorised as negligible risk, 32% low, 65% medium, 3% high and 0% very high risk. Such qualitative risk-based tools can assist the policy community not only to target high risk areas, but also to develop mitigation strategies that are sensitive to the different ways in which risk is produced. Capacity for long-term cross-disciplinary research is argued to be the means by which these integrated and more sustainable solutions may emerge.