Background: Acute diarrhea is defined as the passage of stools with abnormal consistency and frequency in a day (e.g. more than three times) which lasts for less than two weeks. Diarrheal diseases are reported as the leading cause of mortality among children aged five years and below. Stool or blood culture tests are very important to determine proper management for diarrheal illness. Objective: to investigate the difference between the role of stool and blood cultures in the diagnosis of acute diarrheal disease and to determine the predictors of positive cultures in patients with diarrheal illness. Subjects and Methods: This prospective cohort study included 77 children. Forty-one of them were males and 36 were females with acute diarrhea with ages between 6months to 5 years who attending Al-Azhar Assiut University Hospital starting from May 2018 until May 2019. Results: The results of the present study demonstrated that Stool culture was positive in 41 cases and blood culture was positive in 8 cases. All cases with positive blood culture were also had a positive stool culture. Twenty-one cases were positive for campylobacter in stool culture and 2 cases of them were pseudomonas positive in blood culture. Twelve cases were salmonella positive in stool culture and 2 cases of them were pseudomonas positive in blood culture. Seven cases were E-coli positive in stool culture and 2 cases of them were E-coli positive and 1cases was positive for pseudomonas in blood culture. Conclusion: The results of this study revealed that stool or blood culture tests are very important to determine proper management for diarrheal illness, Stool culture is more sensitive than blood culture and in severely ill patients, blood culture was required.