Diarrheal disease is the second most frequent cause of mortality in children younger than 5 years worldwide, causing more than half a million deaths each year. Our knowledge of the epidemiology of potentially pathogenic agents found in children suffering from diarrhea in sub-Saharan African countries is still patchy, and thereby hinders implementation of effective preventative interventions. The lack of cheap, easy-to-use diagnostic tools leads to mostly symptomatic and empirical case management. An observational study with a total of 241 participants was conducted from February 2017 to August 2018 among children younger than 5 years with diarrhea in Lambaréné, Gabon. Clinical and demographic data were recorded, and a stool sample was collected. The samples were examined using a commercial rapid immunoassay to detect Rotavirus/adenovirus, conventional bacterial culture for Salmonella spp., and multiplex real-time PCR for Cryptosporidium spp., Giardia lamblia, Cyclospora cayetanensis, enterotoxigenic Escherichia coli (ETEC), and enteroinvasive Escherichia coli (EIEC)/Shigella. At least one infectious agent was present in 121 of 241 (50%) samples. The most frequently isolated pathogens were EIEC/Shigella and ETEC (54/179; 30.2% and 44/179; 24.6%, respectively), followed by G. lamblia (33/241; 13.7%), Cryptosporidium spp. (31/241; 12.9%), and Rotavirus (23/241; 9.5%). Coinfection with multiple pathogens was observed in 33% (40/121) of the positive cases with EIEC/Shigella, ETEC, and Cryptosporidium spp. most frequently identified. Our results provide new insight into the possible causes of diarrheal disease in the Moyen-Ogooué region of Gabon and motivate further research on possible modes of infection and targeted preventive measures.