Enteroaggregative Escherichia coli (EAEC) is commonly associated with pediatric diarrhea, in developing countries.. In this study, we investigated the distribution of virulence genes, enterotoxin and biofilm formation among EAEC strains isolated from stools of children with diarrhea referred to three hospitals in south east Iran..Patients and A total of 464 diarrheic stools were screen for the presence of E. coli using conventional tests. Well isolated colonies were then evaluated for the presence of EAEC diagnostic genes (aggR and pCVD432) by duplex polymerase chain reaction (D-PCR). Positive samples were further subjected to three sets of multiplex-PCR for detection of fimbrial subunits (AAF), serine protease autotransporter toxins (SPATE) and at least one enterotoxin gene. Hemolytic activity was observed on sheep blood agar. Biofilm formation was measured by using a microtiter plate assay.. Among the 322 E. coli isolated, 23 were identified as EAEC. All EAEC carried a 630-bp fragment of the plasmid (pAA) encoded pCVD432 and aggR genes. Four major EAEC fimbriae variants aggA, agg4A, agg3A and aafA were detected, with the frequencies of 21.7% (n = 5), 26.8% (n = 6), 21.7% (n = 5) and 4.3% (n = 1), respectively. The class I and II virulence toxins pic, sat, sepA, pet and sigA were detected with frequencies of 56.5% (n = 13), 30.4% (n = 7), 26.8% (n = 6), 21.7% (n = 5) and 4.3% (n = 1), respectively. A heat-stable Shigella enterotoxin-1 gene (astA) was detected in 17.3% (n = 4) of the cases. In addition, 56.4% of the EAEC isolates were α-hemolytic. Quantification of the biofilm revealed six isolates with strong biofilm.. Overall, except for pCVD432 and aggR, we detected high heterogeneity of virulence factors among EAEC isolates causing diarrhea in children. One set of genes, in the combination, pic- sepA- agg4A, was associated with strong biofilm..