Background Nucleic acid-based molecular techniques in current laboratory practice allow the identification of a broad range of respiratory microorganisms. However, due to asymptomatic carriage, the detection of microorganisms in the respiratory tract does not necessarily indicate disease. The study aimed to investigate the prevalence of the different viruses that colonize the airways and their association with the occurrence of either upper respiratory tract infection (AURTI) or lower respiratory tract infection (ALRTI) in children. Methods A matched case-case-control study included ALRTI cases, AURTI cases, and healthy controls and was conducted at Kunming Children’s Hospital. Oropharyngeal swabs were collected for eight viral pathogens and analyzed by probe-based multiplex RT-PCR. The association of each pathogen with disease status was determined by comparing the results between each participant group. Results From March 1, 2021 through February 28, 2022, 278 participants in each group were investigated. Viral infection was detected in 53.96%, 37.05%, and 12.23% of the ALRTI cases, AURTI cases, and healthy controls, respectively. Human respiratory syncytial virus (RSV), adenovirus (ADV), and parainfluenza virus-3 (PIV-3) were the most frequently documented viruses. RSV/ADV was the most frequent combination detected in coinfection. RSV was independently associated with both ALRTI and AURTI. Additionally, PIV-3 was only relevant to ALRTI. Conclusions RSV and PIV-3 were causes of either ALRTI or AURTI in pediatric patients in Kunming. These results provide initial evidence of the potential of microbiota-based diagnostics for the differential diagnosis of severe acute respiratory infections using primary site samples.