Objective Acute respiratory infections are a major global public health concern. However, there are few epidemiological studies investigating pathogens associated with respiratory tract infections in Guizhou Province, China. Methods We collected 17,850 blood samples from Guizhou Provincial People’s Hospital between November 2018 and May 2023 to investigate the epidemiological characteristics of respiratory pathogens and their spread during the SARS-CoV-2 epidemic in Guizhou Province. Results We identified influenza virus and Mycoplasma pneumoniae as the predominant pathogens involved in acute respiratory infections in the study area. Immunoglobulin M positivity for respiratory syncytial virus, influenza virus, and M. pneumoniae showed a strong correlation with the clinical diagnosis of pneumonia. Seasonal epidemic patterns were observed for influenza A and B viruses. Following the SARS-CoV-2 outbreak, there was a significant decrease in the positive rates for most respiratory pathogens, particularly influenza A and B, Legionella pneumophila, and respiratory syncytial virus. Conclusion This retrospective study contributes to the epidemiological evidence regarding respiratory pathogens in Guizhou Province, thereby enhancing the surveillance network for respiratory pathogens in China and providing valuable guidance for local hospitals.