The aim of this study was to compare the virulence characteristics and phylogenetic features of enteroaggregative Escherichia coli (EAEC) strains from adults with and without diarrhoea and to search for associations between the analysed genes and carrier or diarrhoeagenic strains, respectively. Faecal samples of 487 healthy humans were screened for EAEC strains and compared with isolates from diarrhoeal patients. Virulence and virulence-associated gene typing, serotyping, multilocus sequence typing and antibiotic susceptibility testing were performed for characterization of the isolates. Characteristics significantly linked to carrier strains or to diarrhoeagenic strains were determined. From 487 stool samples, 24 EAEC strains were obtained. Comparison with strains originating from diseased persons showed a statistically significant association of the genes sat (P = 0.002) and agg3C (P = 0.0139) with the carrier strains, and of pCVD432 (P = 0.0001), aap (P = 0.003), aggR (P = 0.0048) and air (P = 0.031) with the diarrhoeagenic strains. Our study indicates that a certain subset of EAEC is unrelated to diarrhoea, for which sat and agg3C may be markers. Our results further suggest that diarrhoeagenic EAEC strains are distinguishable from carrier strains and suggest that, in addition to well-established markers such as pCVD432 and aggR, aap and air may be useful additional markers to define EAEC as an aetiological agent of diarrhoea in adults.