Background: Acute gastroenteritis is an important public health problem all over the world representing a great economic burden especially in the developing countries. Escherichia coli (E. coli) represents about 30% of bacterial causes especially serotype O157:H7. Infected patients may undergo severe complications in the form of dehydration and hemolytic uremic syndrome (HUS). WHO Global Priority Pathogens (GPP) List reported E. coli O157:H7 as a critical pathogen requiring regular monitoring and surveillance. Objectives: The purpose of this cross sectional study is to highlight the occurrence and determinants factors of this serotype isolated from diarrheal cases in Beni-Suef Governate. Frequent monitoring of foodborne bacteria is essential as they may act as a reservoir carrying and transferring resistance genes to humans. Multidrug resistance (MDR) transmission in this manner would make human bacterial infection is very serious and difficult to be treated. Methodology: The study was carried out on a total 457 cases randomly attended to different hospitals of Beni-Suef Governate (Internal Medicine Outpatient Clinic and Emergency Department) over a period of 2 months. Clinically diagnosed patients with gastroenteritis were included. E. coli O157:H7 was identified by API 20E test kit (bioMérieux, France) and serotyped using latex agglutination test (Oxoid, UK). Sociodemographic factors for cases were also assessed. Results: E. coli O157:H7 was isolated with a prevalence rate 11% (41 out of 457) ; aged from 4- 33 years old; (68.3%) males, (51.2%) urban residence and most patients had a habit of outdoor food consumption (70.7%) and a direct contact with animals (82.9%). About 25 cases (61%) needed admission, 10 cases (40%) had moderate dehydration, (13 cases, 52%) had HUS ended with a surviving fate and 2 cases (8%) had HUS ended with death representing the fatality rate. Self-limited gastroenteritis was associated with watery diarrhea with a statistically significant difference; p. value= 0.014. Age among cases who died was lower than survived cases with a significant difference; p. value= 0.006 and all dead patients were children with a statistically significant difference p. value= 0.036. There was no a statistically significant difference regarding sex and residence compared to the fate of admission (p-value> 0.05). Conclusion: E. coli O157:H7 serotype was isolated from Beni-Suef Governate with a relatively high prevalence during the period of the study. Shortage of laboratory facilities to diagnose such pathogen interferes with its definitive diagnosis with subsequent complicated fate for infected cases. Proper handling practices as well as public awareness about the epidemiology of the pathogen should be settled. Vehicles of transmission such as food products and water should be decontaminated to prevent the infection.