There are an estimated 17 million human diarrhoea cases annually in the United Kingdom. In 2008 and 2009, enteroaggregative E. coli (EAEC) were identified in 1.9% of stools. However, it remains unclear whether there is a causal link between presence of EAEC and disease. This study used bacterial load, the presence of co-infections and demographic data to assess if EAEC was independently associated with intestinal infectious disease. Quantitative real-time PCR data (Ct values) generated directly from stool specimens for several pathogen targets were analysed to identify multiple pathogens, including EAEC, in the stools of cases and healthy controls. Sensitivity and specificity using Ct value (60% and 60%) was not useful for identifying cases or controls, but an independent association between disease and EAEC presence was demonstrated: multivariate logistic regression for EAEC presence (odds ratio: 2.41; 95% confidence interval: 1.78–3.26; p<0.001). The population-attributable fraction was 3.3%. The group of bacteria known as EAEC are associated with gastrointestinal disease in at least half of the cases with EAEC positive stools. We conclude that the current definition of EAEC, by plasmid gene detection, includes true pathogens as well as non-pathogenic variants.