Abstract: OBJECTIVE To explore the clinical application value of metagenomic next-generation sequencing (mNGS) in etiological diagnosis of bone infections after exposure to short-term empirical antimicrobial drugs. METHODS A total of 35 eligible patients with bone infections who were treated in orthopedics department of Ji'an Central People's Hospital from Jan. 2022 to Dec. 2023 were enrolled in the study. The matched lesion specimens were respectively collected before the use of antibiotics (T0) and after the empirical antimicrobial therapy for 3 to 7 days (T1). The traditional microbial culture and mNGS were conducted simultaneously. The positive rates, isolation rates of pathogens and reporting time were observed and compared based on composite clinical reference standards between the two methods at different time points. RESULTS The positive rate of mNGS was 82.86%(29/35)at T0, higher than 31.43%(11/35)of the traditional culture; the positive rate of mNGS was 80.00%(28/35) at T1, higher than 28.57%(10/35) of the traditional culture. It was suggested that mNGS maintained high level of etiological detection among the bone infection patients who received empirical antimicrobial therapy. Totally 40 strains of pathogenic microorganisms were detected by mNGS, and 12 strains were detected by traditional culture. 5 cases of mixed infections and 2 cases of infections with rare pathogens were additionally identified by mNGS, including 1 case infected with Brucella and 1 case infected with Mycobacterium tuberculosis composite group. The median reporting time of the mNGS was 26.50 h, shorter than 96.70 h of the traditional culture(P＜0.001). CONCLUSIONS mNGS exhibits higher positive rate and shorter reporting time than the traditional culture in the etiological diagnosis of bone infections. As a major detection technique, mNGS can be used for the patients who have received short-term empirical antimicrobial therapy, are negative for traditional culture, or have suspected mixed infections, infections with rare pathogens.