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This study evaluated the microbiological quality and prevalence of Escherichia coli and diarrheagenic E. coli pathotypes (DEP) among fresh-squeezed orange juice from street vendors and their hygienic practices. Sixty orange juice stalls in Mexico City were visited, and 60 samples were purchased. Most (65%) of the street vendors had poor hygienic practices. Twelve (20%) prepared orange juice at the time of purchase, while the other stalls sold already-squeezed orange juice. All samples analyzed were positive for aerobic mesophilic bacteria, with limits ranging from 1 to 6.8 log CFU/mL. A total of 35 (58.3%), 27 (45%), and 5 (8.3%) samples were positive for total coliforms, fecal coliforms, and E. coli, respectively, present at concentrations ranging from < 3 to > 1100, < 3 to > 1100, and < 3 to 53 MPN/mL, respectively. Two samples harbored DEP at concentrations of 3.6 and 9.2 MPN/mL. Both samples harbored Shiga toxin-producing E. coli strains, positive for the Shiga toxin 2 (stx2) locus. Our results suggest that consumption of fresh-squeezed street-vended orange juice may pose a health risk for consumers, revealing the necessity to implement adequate hygiene and safety practices to protect the health of consumers.