Prompt pathogen identification can have a substantial impact on the optimization of antimicrobial treatment. The objective of the study was to assess the diagnostic value of next-generation sequencing (NGS) for identifying pathogen and its clinical impact on antimicrobial intervention in immunocompromised patients with suspected infections.This was a retrospective study. Between January and August 2020, 47 adult immunocompromised patients underwent NGS testing under the following clinical conditions: 1) prolonged fever and negative conventional cultures; 2) new-onset fever despite empiric antimicrobial treatment; and 3) afebrile with suspected infections on imaging. Clinical data, including conventional microbial test results and antimicrobial treatment before and after NGS, were collected. Data were analyzed according to documented changes in antimicrobial treatment (escalated, no change, or de-escalated) after the NGS results.The median time from hospitalization to NGS sampling was 19 d. Clinically relevant pathogens were detected via NGS in 61.7% of patients (29/47), more than half of whom suffered from fungemia (n=17), resulting in an antimicrobial escalation in 53.2% of patients (25/47) and antimicrobial de-escalation in 0.2% of patients (1/47). Antimicrobial changes were mostly due to the identification of fastidious organisms such as Legionella, Pneumocystis jirovecii, and Candida. In the remaining three cases, NGS detected clinically relevant pathogens also detected by conventional cultures a few days later. The antimicrobial treatment was subsequently adjusted according to the susceptibility test results. Overall, NGS changed antimicrobial management in 55.3% (26/47) of patientst, and conventional culture detected clinically relevant pathogens in only 14.9% of patients (7/47).With its rapid identification and high sensitivity, NGS could be a promising tool for identifying relevant pathogens and enabling rapid appropriate treatment in immunocompromised patients with suspected infections.Copyright: © World Journal of Emergency Medicine.