Background:　The　objective　of　this　study　is　to　utilize　metagenomic　next-generation　sequencing　(mNGS)　to　analyze　the　risk　factors　causing　negative　microbial　cultures,　comprehensively　delineate　the　microbial　profiles　neglected　by　traditional　cultures,　and　optimize　the　pathogenetic　diagnostic　procedure　accordingly.　Research　design　and　methods:　We　enrolled　341　patients　diagnosed　with　OI　at　our　center　between　2016　and　2022,　and　gathered　data　including　age,　gender,　clinical　diagnosis,　duration　of　antibiotic　use　prior　to　sampling,　microbial　culture　results,　and　mNGS　results　for　these　patients.　According　to　microbial　detection　results,　risk　factors　for　negative　microbial　culture　and　mNGS　results　were　investigated　through　univariate　and　multivariate　analyses,　and　the　microbial　profile　in　cases　with　negative　microbial　cultures　was　summarized　in　conjunction　with　mNGS　results.　Building　upon　this,　we　suggest　strategies　to　enhance　the　positivity　rate　of　microbial　cultures　based　on　clinical　experience.　Results:　Invasive　osteoarticular　infection　(IOI),　multi-infections,　rare　pathogen　infections,　and　prior　antibiotic　use　are　risk　factors　for　negative　microbial　cultures.　When　the　duration　of　prior　antibiotic　use　is　≥3　days,　mNGS　demonstrates　significantly　higher　pathogen　detection　efficiency　than　microbial　culture.　Moreover,　the　risk　of　negative　microbial　culture　increases　by　4.8　times　with　the　exposure　to　each　additional　risk　factor　(OR=4.043,　95%CI　[2.835,　5.765],　P＜0.001).　Additionally,　over　one-third　of　culture-negative　OI　involve　polymicrobial　infections　or　rare　pathogens.　Conclusions:　Clinicians　should　tailor　microbial　culture　strategies　based　on　patient　conditions.　When　needed,　they　can　collaborate　with　mNGS　or　optimize　microbial　culture　conditions　based　on　mNGS　results　to　enhance　the　efficiency　of　pathogen　diagnosis.　Copyright　©　2024　Ding,　Huang,　Lin,　Chen,　Wang,　Li,　Huang,　Fang　and　Zhang.