Background: Diarrhea is the most frequent health problem among children in developing countries. This study investigated the bacterial and viral etiology and related clinical and epidemiological factors in children with acute diarrhea in Ouagadougou, Burkina Faso. Methods: Stool specimens were collected from 283 children under 5 years of age visiting hospital due to acute diarrhea and from 60 healthy controls of similar age. Pathogens were investigated by using conventional culture techniques, PCR and immunochromatographic testing. Salmonella and Shigella strains were serotyped and their susceptibility to 23 antimicrobial agents was determined by the agar dilution method. Results: At least one pathogen was detected in 64% of the 283 patients and in 8% of the 60 controls (p < 0.001). Rotavirus was found in 30% of the patients, followed by diarrheagenic Escherichia coli (24%), Salmonella enterica ssp. enterica (9%), Shigella spp. (6%), adenovirus (5%) and Campylobacter spp. (2%). Multiple pathogens were found in 11% of the patients and in 2% of the controls (p = 0.028). Viruses were found mainly in children of ≤ 2 years of age, whereas bacteria were equally prevalent among all the age groups. Viral infections occurred mostly during the cool dry season and the bacterial infections during the rainy season. Fever (64%) and vomiting (61%) were the most common symptoms associated with diarrhea. Only one Salmonella strain was resistant to nalidixic acid and ciprofloxacin. Of the Shigella strains, one was resistant to nalidixic acid but 81% to trimethoprim- sulfamethoxazole, 63% to streptomycin and 50% to ampicillin. Most of all the other Salmonella and Shigella strains were sensitive to all antimicrobials tested. Conclusion: Rotaviruses and diarrheal E. coli were the most predominant pathogens associated with acute diarrhea in Burkinabe children. Constant antimicrobial surveillance is warranted to observe for the emergence of enteric bacteria resistant to antimicrobials that are important in treatment also of severe infections.