Objectives <p>This study aimed to evaluate the diagnostic performance of gene-based bioinformatic analysis via next-generation sequencing (NGS) compared to conventional culture and microscopy in identifying microorganisms and antibiotic resistances in odontogenic abscesses. Additionally, the influence of selected clinical indicators including prior antibiotic therapy, in-hospital antibiotic treatment, and detection of antibiotic resistance on length of stay (LOS) was analysed.</p> Materials and methods <p>In patients undergoing extraoral incision and drainage of odontogenic abscesses at the Department of Oral and Maxillofacial Plastic Surgery, University Hospital Bonn, two microbiological swabs (NGS and conventional) were taken intraoperatively from each patient. Microbial profiles and resistance patterns were assessed and correlated with clinical data.</p> Results <p>Bacteria were identified in 100% of NGS swabs compared to 68.1% in conventional swabs (<i>p</i> &lt; 0.001). NGS detected a median of 8 bacterial genera per sample versus 1 via conventional methods, primarily due to superior detection of anaerobes (median 7 vs. 0). Both methods equally identified aerobic genera (median 1 each). Fungal organisms and antibiotic resistances were also more frequently detected using NGS (<i>p</i> &lt; 0.001). Prior antibiotic therapy was associated with a significantly reduced LOS (<i>p</i> = 0.030). Neither the type of in-hospital antibiotic regimen nor the presence of resistance influenced LOS.</p> Conclusions <p>NGS substantially enhances the detection of polymicrobial communities, including anaerobes and fungi, and identifies antibiotic resistances more effectively than conventional diagnostics in odontogenic abscesses.</p> Clinical Relevance <p>NGS offers rapid, comprehensive pathogen profiling and resistance testing, supporting quick establishment of tailored antimicrobial therapy.</p>