Despite its occurence as a commensal in the human intestine, Escherichia coli is also known as a versatile gastrointestinal pathogen. Identification of diarrheagenic E. coli (DEC) requires the accurate discrimination of pathogenic strains from commensal flora, and this is not an easy task if the diagnostic tools are inadequate. As the information regarding the relative contribution of DEC among other identifiable causes of infectious diarrhea in Romanian patients is scarce, a prospective study was conducted to evaluate the prevalence of enteropathogenic Escherichia coli (EPEC) and verotoxin-producing Escherichia coli (VTEC) isolates in the diarrheagenic stool specimens of 120 children and 270 adults. PCR-based detection of the eae, bfp, vtx1 and vtx2 genes was added to the conventional culture and slide agglutination with 12 commercial EPEC antisera and O157:H7 antisera for identifying EPEC and VTEC isolates. Even though E. coli colonies belonging to traditional EPEC serogroups were isolated from 35 children and 17 adults, only the isolates recovered from 16 children and 2 adults harboured at least one of the targeted pathogenicity-associated genes. The children shedding EPEC outnumbered the adults (16.7% vs. 7.4%). Based on the virulence genotype identified, the prevalence of atypical EPEC (eae+) was higher than of typical EPEC (eae+ bfpA+), and typical EPEC identification was restricted to children. Owing to the molecular analysis 5 children and 10 adults that could have been overlooked by the routine microbiological investigation were diagnosed as infected with VTEC. Considering that none of the screened stool specimens was positive for E. coli O157:H7, this study reports for the first time the presence of VTEC nonO157 in local patients with diarrhea. Our results bring evidence that both EPEC and VTEC isolates are circulating as agents of local sporadic cases of human diarrhea. Further studies are needed to evaluate the contribution of DEC to the human disease burden in Romania, based on improved diagnostic tools targeting the main virulence traits of E. coli clinical isolates.