Objective To investigate the clinical application value of metagenomic next-generation sequencing (mNGS) in the etiological diagnosis of bone and joint infection. Methods Clinical data of patients with suspected bone and joint infection in a hospital in Guangdong Province from January 2021 to June 2022 were retrospectively ana- lyzed. Specimens from infected sites of bone and joint infection of all patients were taken for mNGS detection and microbial culture, infected sites of bone and joint infection were performed CT imaging examination. The efficacy of the three methods in diagnosing bone and joint infection was compared. Results A total of 108 suspected patients with bone and joint infections were included in the study, and 126 specimens were taken from bone and joint infection site. The sensitivity of mNGS detection was 84.38%, higher than that of microbial culture (48.44%) and CT imaging examination (62.50%), with statistically significant difference (P < 0.001). The negative predictive value of mNGS detection was 85.51%, higher than that of microbial culture (64.89%) and CT imaging (68.42%); The specificity of microbial culture was 98.39%, higher than that of mNGS detection (95.16%) and CT imaging (83.87%); the positive predictive value of microbial culture was 96.88%, higher than that of mNGS detection (94.74%) and CT imaging (81.63%), differences were all statistically significant (P < 0.05). Compared with microbial culture, mNGS could detect more pathogens, especially Mycobacterium tuberculosis (9.52% VS 2.38%; χ²=5.742, P < 0.05). The concordance rates between pre- and post-host removal mNGS sequencing results and clinical diagnosis results of 56 specimens with high human background were 87.50% (49/56) and 78.57% (44/56), respectively, with no statistically significant difference (P=0.125). Conclusion The sensitivity of mNGS in diagnosing pathogens of bone and joint infections is high, with a shorter detection period and the ability to detect more pathogens, which has important clinical value.