We used a multiplex polymerase chain reaction (PCR) and a quantitative real-time PCR to determine the distribution of three enteroaggregative Escherichia coli (EAEC) virulence-related genes in stool samples from hospital patients and school children in the Venda region of South Africa. At least one gene was found in 52 (16.5%) samples, 50 (19.6%) from hospitals and 2 (3%) from schools. The AA probe was found in 36 (69%), the aggR gene was found in 41 (79%), and the aap gene was found in 49 (94%) of all positive samples. EAEC was significantly associated with diarrhea and intestinal inflammation and was significantly higher (χ2 = 5.360, P = 0.021) in human immunodeficiency virus (HIV)-positive persons (29.5%) than in HIV-negative persons (13.7%). The presence of EAEC genes was significantly associated with occult blood (χ2 = 30.543, P < 0.0001) in the stool samples. This study suggests that the clinical presentation of EAEC infection may be directly related to the bacterial load as well as to the genetic characteristics of the strains involved.