Background and Objectives: Gastroenteritis is a leading cause of childhood morbidity and mortality in developing countries. Our aim was to determine the prevalence of bacterial, viral and parasitic aetiology of gastroenteritis in children aged < 5 years in the Najran region, southwestern Saudi Arabia, to determine the contribution of these enteropathogens in childhood diarrhoeal diseases and to put forward effective preventive measures for controlling the disease in the future. Design and Setting: A descriptive study conducted at Najran Maternity and Children's Hospital and the Microbiology Department of the Najran University College of Applied Medical Sciences from October 2011 to June 2012. Patients and Methods: Stool samples were collected from 326 children aged ≤ 5 years with diarrhoea and examined for bacterial, viral and parasitic enteropathogens. Results: Seventy-two (22.1%) samples were positive for viral pathogens, including 56 (17.2%), 12 (3.7%) and 4 (1.2%) samples for rotavirus, adenovirus and astrovirus, respectively. Thirty-five (10.7%) samples were positive for bacterial pathogens, including 28 (8.6%) and 7 (2.1%) samples for Salmonella and Shigella spp. isolates, respectively. Pathogenic parasites were detected in only 4 (1.2%) samples, including 3 (0.9%) Giardia lamblia and 1 (0.3%) Entamoeba histolytica isolates. Conclusions: Rotavirus is the most common pathogen in paediatric acute gastroenteritis in the Najran region. This fact, in combination with the severity of the infections, warrants consideration of a rotavirus vaccine in the childhood immunization program in Saudi Arabia. On the basis of clinical and laboratory parameters, it appears possible to distinguish between the different causative agents of acute gastroenteritis.