Metagenomic next-generation sequencing (mNGS) is a new high-throughput sequencing method that may have great importance in early diagnosis and clinical management of sepsis. This study aimed to detect the difference between mNGS and comprehensive routine microbiological test (CMT), and to explore the diagnostic efficacy of mNGS in septic patients. This study retrospectively analyzed 150 sepsis patients who were admitted to the intensive care units of 4 hospitals in Southwest China from October 1, 2018, to October 1, 2021, and underwent both blood mNGS and CMT. The demographic and clinical characteristics of the patients were recorded, and the distribution of pathogens was analyzed. Additionally, the diagnostic performance and concordance between mNGS and CMT were compared to evaluate the etiological diagnostic value of mNGS in sepsis patients. In this study of 150 sepsis patients, bacterial infections were identified in 126 (84.0%), viral in 15 (10.0%), and fungal in 9 (6.0%). Among the sample types, sputum was most common, representing 62% of the total cases. Bronchoalveolar lavage fluid constituted 58.7%, blood 56.0%, with other specimens including pleural fluid at 29.3%, pus at 19.3%, swabs at 9.3%, cerebrospinal fluid at 8.7%, tissue at 6.0%, and bone marrow at 5.3%. mNGS demonstrated a diagnostic accuracy of 56.0% for sepsis, with a sensitivity of 84.4%, specificity of 26.0%, a positive predictive value of 54.6%, a negative predictive value of 61.3%. Metagenomic testing enables the rapid and early identification of infectious pathogens in sepsis patients, especially fungi and viruses. The study found that mNGS has high sensitivity in diagnosing sepsis patients, particularly for fungal and viral infections. mNGS technology is beneficial for critically ill sepsis patients.Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.