This study aimed to analyze the pathogenic bacteria spectrum in invasive and primary osteoarticular infection (IOI and POI) and compare the pathogen detection rate of metagenomic next-generation sequencing (mNGS) and microbial culture in IOI and POI. The suspected POI and IOI cases from 2014-2021 were included. The diagnosis of POI or IOI was made by at least two orthopedic surgeons, two infectious diseases specialists, and one senior microbiologist. Demographic characteristics, microbial culture results, and so on were recorded. The pathogenic bacteria spectrum in IOI and POI were analyzed, and the ability of mNGS and microbial culture in pathogen detection in IOI and POI were compared. There were 52 POI cases and 92 IOI cases; the common pathogen in POI and IOI were both Staphylococcus aureus. There are more cases with negative microbial culture results and multiple infections in IOI, and many cases were caused by rare and fastidious bacteria. The introduction of the mNGS could significantly increase the pathogen detection rate to 92.39% in IOI, which was 8.69% higher than that of microbial culture (P = 0.007), whereas the improvement in POI was limited to about 2%. mNGS is an promising tool for IOI pathogen detection.