OBJECTIVE To investigate the virulence factors including hemolysin production, β-lactamase production, and biofilm formation. Antimicrobial resistance and plasmid content of 20 Escherichia coli isolates obtained from feces and Tigris water were screened. METHODS Ten clinical and ten environmental E. coli isolates were collected from children diarrhea and swim areas on Tigris River in Baghdad city, Iraq, respectively. The bacterial isolates were identified by cultural characteristics, Gram stain, biochemical tests, and screened for the presence of E. coli O157:H7 serotype. Bacterial E. coli isolates were investigated for hemolysin production, biofilm formation, and β-lactamase production. Antibiotics susceptibility and plasmid content were determined. RESULTS A total of ten clinical and ten water E. coli isolates were studied. Results showed that all E. coli isolates give negative results for latex O157:H7. Virulence factors analysis showed that 6/10 water isolates and 2/10 clinical isolates were hemolytic, 5/10 water isolates and 3/10 clinical isolates were biofilm formation, and 7/10 water isolates and 4/10 clinical isolates were β-lactamase producer. Antibiotics profile showed that all bacterial isolates were multidrug resistant. All E. coli isolates (100%) were resistant to carbenicillin, cefodizime, imipenem, and piperacillin. The plasmid DNA analysis showed that all E. coli isolates contained plasmid with molecular weight range between 4.507 kbp and 5.07 kbp, but clinical isolates contained multiple small and mega plasmids. CONCLUSIONS Our study revealed that E. coli isolates from river water exhibit a higher level of hemolysin production, β-lactamase production, and biofilm formation than feces isolates may be due to long adaptation. On the other hand, clinical E. coli isolates from feces showed higher level of antibiotic resistance and have multiple plasmids.