Abstract Background Diagnosing agents causing respiratory infections remains a global challenge due to the numerous coexisting agents and similar symptoms. This study evaluates the epidemiology of viral and bacterial agents responsible for major respiratory infections through molecular detection in patients across various Brazilian hospital units served by a clinical diagnostic and health laboratory. Understanding the distribution and prevalence of these pathogens is crucial for effective diagnosis, treatment, and prevention strategies in the face of evolving epidemiological landscapes. Methods Utilizing an internal database, we conducted a comprehensive analysis of tests performed on the FilmArray® system. This system employs multiplex PCR technology, capable of simultaneously identifying 21 respiratory agents in a single sample obtained from nasopharyngeal and bronchial lavage specimens collected from diverse geographical regions across Brazil. The data for the examination of patient samples with respect to gender, age group, geographical regions, and the prevalence of various agents between January 2021 and April 2023 were retrieved from the database and analyzed. To ensure privacy, sensitive and individual data from all patients were meticulously preserved throughout the study. Results The analysis encompassed a diverse sample of 477 patients, originating from 26 cities across 11 Brazilian states, including Goiás, Minas Gerais, Mato Grosso, Pará, Paraná, Rio de Janeiro, São Paulo, Tocantins, Amazonas, Bahia, and Distrito Federal. The distribution of patients was nearly split between hospital settings (39.8%) and outpatient units, with hospital patients predominantly from departments such as internal medicine, cardiology, and nephrology. In contrast, outpatient samples were primarily requested by specialists in pulmonology and infectious diseases. The demographic profile revealed an average age of 36 years, with a majority male representation (54%). However, a notable shift occurred in the first four months of 2023, where females constituted 60.8% of the sample. The overall test positivity rate stood at 42.3%, with a significant detection of 71% (15 out of 21) of the surveyed respiratory agents at some point during the study period. The most frequently identified pathogens were Rhinovirus, Enterovirus, and SARS-CoV-2, collectively accounting for 55.1% of the positive findings. Additionally, mixed infections were observed in 29 (6%) of the patients, with combinations such as SARS-CoV-2 and Rhinovirus, SARS-CoV-2 and H3N2, and SARS-CoV-2 and RSV, among others, highlighting the complexity of respiratory infections. Conclusions The findings from this study underscore the critical role of advanced molecular diagnostics in the Brazilian clinical-laboratory landscape. The utilization of multiplex PCR technology has proven to be a pivotal tool in accurately identifying a wide array of respiratory pathogens, thereby enhancing the precision of diagnosis and treatment strategies. The detection of a high prevalence of agents such as Rhinovirus, Enterovirus, and SARS-CoV-2, along with the identification of mixed infections, highlights the complexity of respiratory infections and the necessity for robust diagnostic tools. This study not only contributes to the understanding of the epidemiology of respiratory infections in Brazil but also emphasizes the importance of continuous advancements in molecular diagnostics for better patient care and public health management.