To explore the application value of the second-generation metagenomic next-generation sequencing (mNGS) in the detection of pathogens in patients with pulmonary infection.We conducted a retrospective analysis of 65 pulmonary infection cases treated at our institution and the Fifth People's Hospital of Shanghai between January 2021 and May 2023. All subjects were subjected to mNGS, targeted next-generation sequencing (tNGS), and conventional microbiological culture. A comparative analysis was performed to evaluate the diversity and quantity of pathogens identified by these methodologies and to appraise their respective diagnostic capabilities in pulmonary infection diagnostics.The mNGS successfully identified etiological agents in 60 of the 65 cases, compared to tNGS, which yielded positive results in 42 cases, and conventional laboratory cultures, which detected pathogens in 24 cases. At the bacterial genus level, mNGS discerned 9 genera, 11 species, and 92 isolates of pathogenic bacteria, whereas tNGS identified 8 genera, 8 species, and 71 isolates. Conventional methods were less sensitive, detecting only 6 genera, 7 species, and 33 isolates. In terms of fungal detection, mNGS identified 4 fungal species, tNGS detected 4 isolates of the Candida genus, and conventional methods identified 2 isolates of the same genus. Viral detection at the species level revealed 10 species and 46 isolates by mNGS, whereas tNGS detected only 3 species and 7 isolates. The area under the receiver operating characteristic curve (AUC) with 95% confidence intervals for diagnosing pulmonary infections was 0.818 (0.671 to 0.966) for mNGS, 0.668 (0.475 to 0.860) for tNGS, and 0.721 (0.545 to 0.897) for conventional culture.The mNGS demonstrates superior diagnostic efficacy and pathogen detection breadth in critically ill patients with respiratory infections, offering a significant advantage by reducing the time to diagnosis. The enhanced sensitivity and comprehensive pathogen profiling of mNGS underscore its potential as a leading diagnostic tool in clinical microbiology.Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.