Background: Exudative diarrhea is a significant global public health issue, particularly affecting children under the age of 5. Identifying the cause of diarrhea is crucial for epidemiological surveillance and, in some cases, for ensuring appropriate treatment for patients. Objectives: The aim of this study was to investigate the frequency of bacteria responsible for exudative diarrhea in samples from children under five years old in a tertiary hospital in Iran. Methods: In this multicenter cross-sectional descriptive study, 104 children with exudative diarrhea who were referred to Mofid Children's Hospital in Tehran, as well as hospitals in Hamedan, Ardebil, and Bandar Abbas, from December 2020 to March 2022 were enrolled. DNA extraction was performed using a commercial kit, and the identification of various causative bacteria was conducted through conventional and real-time PCR. Descriptive and inferential statistics were analyzed using SPSS version 22 software. Results: Most of the children with exudative diarrhea were under 12 months old (31%) or between 12 and 24 months old (22%). Boys made up 66% of the participants. Additionally, 70% of the children had a fever, and 58% experienced vomiting. Furthermore, 56% of the patients were dehydrated. The most prevalent bacterial causes of exudative diarrhea were Shigella spp., followed by Salmonella spp., Campylobacter spp., Clostridium difficile, E. coli-Stx1, and E. coli-Stx2. Conclusions: The findings indicated that *Shigella* spp. was the leading cause of diarrhea in children under five years old. The most common signs and symptoms associated with exudative diarrhea were fever and vomiting, which physicians should consider in their diagnostic and treatment processes.