Introduction: Enteroaggregative Escherichia coli (EAEC) is an important agent of acute and persistent diarrhea worldwide. Few cases have been reported in healthy children. EAEC strains are characterized by aggregative adherence (AA) to HEp-2 cells, wherein bacteria are seen in “stacked brick” aggregates attaching to HEp-2 cells and usually to the glass surface between cells. Goal: To identify Enteroaggregative Escherihia coli using multiplex polymerase chain reaction (PCR) and HEp-2 adherence assay in Ulaanbaatar, Mongolia Materials and Methods: A total of 329 E. coli strains were isolated from stool with diarrhea in National Center for Communicable Diseases from July 2012 through September 2014. All specimens were processed by routine microbiological and biochemical tests in the bacteriological laboratories to identify Salmonella spp., Shigella spp. All specimens in our study were negative for these bacterial and parasitic pathogens. The biofilm formation was evaluated by the growth rate of E.coli on plastic surface. PCR assays were used to detect genes of five types of diarrheagenic E.coli (DEC). All of the DEC strains showed mannose-resistant adherence to HEp-2 cells, and aggregative adherence was predominant in these isolates. Bacterial susceptibility to antimicrobial agents determined by the Kirby Bauer disk diffusion method on Muller Hinton agar. Results: EAEC (31.9%) was the most prevalent by PCR and HEp-2 assay comparing with others. EAEC by multiplex PCR in samples (11, 3.3%), followed by enteropathogenic E.coli (EPEC) seen in 2.1%. Enterohemorrhagic E.coli (EHEC) and enteroinvasive E.coli (EIEC) were found in 7 (2.1%) and 1 (0.3%) of the samples. Enterotoxigenic E.coli (ETEC) and diffusely adhering E.coli were detected in 2 (0.6%), respectively. The evaluation of bacterial biofilm formation using 96 well plates showed 309 negative (93%), 15 weak biofilm (4.6%) and 8 moderate biofilm (2.4%) formation for E.coli and no strong biofilm forming strain was detected. Above 50% of antibiotic resistance was observed for ampicillin, trimethoprim/sulfamethoxazole, cefuroxime and cephalotin. Also, 95.4% of isolates were resistant to at least three different classes of antimicrobial agents and considered as multidrug resistance. Conclusion: EAEC is most prevalent pathogen among DEC in our samples. It is necessary to implement EAEC identifying method on Hep-2 assay in our laboratory practice.