Background Diarrhoea is still a major public health issue in developing countries, and it is one of the leading causes of morbidity and mortality in children. We aimed to assess the use of a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay for the detection of five viruses, including rotavirus, norovirus (genogroups 1 and 2), astrovirus, and adenovirus, responsible for gastroenteritis in children under 5 years old in primary care centres in Upper Egypt. Subjects and methods A total of 500 stool samples were collected. Fifty samples were randomly selected for viral examination using multiplex RT-PCR for the detection of rotavirus, norovirus (genogroups 1 and 2), astrovirus, and adenovirus, causing diarrhoea. Results Viruses were detected in 45 (90%) of the 50 stool samples. The most frequently identified virus was norovirus G2, followed by Group A rotavirus, astrovirus and adenovirus. Mixed infection by two and three viruses was observed in 7/50 cases (14%) and 2/50 cases (4%), respectively. Norovirus G1 was not detected in the samples examined. Conclusion Our study reveals that multiplex PCR allows for the detection of multiple viral targets in only one reaction, rendering the assay easier to perform compared to existing testing methodologies (RT-PCR and electron microscopy). Additionally, most of the viruses were detected in summer, and the highest prevalence was in the age group less than 1 year. Norovirus G2 and rotavirus were the most frequent agents and the most common coinfections responsible for gastroenteritis in children.