Abstract: This study investigated the prevalence of Escherichia coli O157:H7 in the stool of confirmed and non-confirmed diarrhoeic HIV/AIDS patients. Escherichia coli O157:H7 was isolated by culture-based and immunomagnetic separation from three hundred and sixty stool swabs. Identification was by conventional IMViC, 20E API and molecular techniques. Confirmed and non-confirmed diarrhoeic HIV/AIDS patients had 56.5% (74/131) and 43.5% (57/131) respectively of E. coli O157:H7. Molecular results indicated that the prevalence of E. coli O157:H7 was 12.16% (9/74) and 8.77% (5/57) from stool swabs of confirmed and non-confirmed diarrhoeic HIV/AIDS patients. Antimicrobial resistance was higher for E. coli O157:H7 isolates from stools of confirmed HIV/AIDS than it was for non-confirmed HIV/AIDS patients. Escherichia coli O157:H7 might be a silent cause of diarrhoea in HIV/AIDS patients. It is recommended that HIV/AIDS patients with diarrhoea should be screened for E. coli O157:H7 and surveillance programmes for these bacteria should be established in both urban and rural areas of South Africa.