Background: Diarrhea can be frequent among Hajj pilgrims; however, data on its etiology are very limited. Patients and methods: A prospective cohort study was conducted among Hajj pilgrims in 2016. Medical follow-up and systematic rectal swabing were performed before leaving France and before leaving Saudi Arabia. Potential pathogens were identified using the BioFire FilmArray (R) Gastrointestinal multiplex qualitative PCR panel. Results: 117 pilgrims were included and 13.7% experienced diarrhea during Hajj. Of the pre-Hajj samples, 32.5% were positive for at least one pathogen compared to 50% of post-Hajj samples (p = 0.0033). Diarrhea associated Escherichia coli. strains, notably enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and Shiga-like toxin-producing E. coli, were acquired by 29.9%, 10.2%, and 6.5% pilgrims, respectively. Pilgrims with resolved diarrhea were significantly more likely to have post-Hajj EAEC positive samples, compared with those who did not suffer diarrhea (55.6% vs 16.5%). We found a lower prevalence of EPEC (22.5%) in pilgrims who declared washing their hands more frequently at the Hajj than usually as compared to others (40.0%). Conclusion: The acquisition of diarrhea associated E coli by Hajj pilgrims is of major concern given the high prevalence rate of third-generation cephalosporin-resistant E. coli in Saudi Arabia.