Diarrhoeagenic Escherichia coli can be considered as the most important etiologic agents of diarrhoea in the Islamic Republic of Iran, particularly in children. This study determined the frequency of diarrhoeagenic E. coli isolates collected from children with acute diarrhoea (n = 50) and a control group (n = 50) at an Iranian referral paediatric centre during a 1-year period. Using multiplex PCR, diarrhoeagenic E. coli was identified in 90% of the case group and 20% of controls. Enterotoxigenic E. coli was the most frequently identified pathotype in both groups (26% in cases; 10% in controls). Shiga toxin-producing E. coli was the second most isolated pathotype (17%), followed by enteroaggregative E. coli (12%). No enteroinvasive E. coli and enteropathogenic E. coli strains were recovered. More than 80% of isolates harboured the fimH gene. This high proportion of diarrhoeagenic E. coli and diversity of E. coli types highlights the need for enhanced surveillance of gastroenteritis agents in children in this country.