Background <p>Pyogenic spondylodiscitis is rarely caused by anaerobic organisms. This retrospective study evaluated the clinical manifestations and treatment outcomes in patients affected by anaerobic spondylodiscitis.</p> Methods <p>We performed a retrospective analysis of medical records from individuals diagnosed with anaerobic spondylodiscitis between January 2018 and January 2024.</p> Results <p>From January 2018 to January 2024, 373 cases of pyogenic spondylodiscitis were identified in our department. Twenty-three patients (6.2%; 16 males and 7 females), with a mean age of 63.7 years (63.7 ± 14.3 years), were diagnosed with anaerobic spondylodiscitis during this period. All patients presented with persistent back pain lasting over one month, 5 patients (21.7%) had fever, and 3 patients (13.0%) had sciatica. The lumbar spine affected 18 patients (78.3%), and the thoracic spine affected 5 (21.7%) patients. Computed tomography images usually reveal erosion and destruction of adjacent endplates and vertebral bodies. T1-weighted Magnetic resonance imaging revealed a reduction in the signal intensity of the vertebral body, endplate and intervertebral disc. T2-weighted images demonstrate increased signal intensity of the vertebral body and/or disc. Cultures of specimens obtained by percutaneous fluoroscopically guided biopsy or surgery were positive for pathogens for 10 (43.5%) of 23 patients, and Next-generation sequencing (NGS) was positive for 19 (95.0%) of 20 patients. The positive rate of NGS is higher than that of cultures (<i>P</i>&lt;0.01). The most common gram-positive coccus is <i>Parvimonas micra</i>, and the gram-negative bacilli is <i>Bacteroid fragilis</i>. One patient (number 1) treated with a 6-week course of antibiotics relapsed 1 month after treatment withdrawal. There was no statistically significant variation in the cure rate between the conservative treatment group and the surgical treatment group (<i>P</i> &gt; 0.05).</p> Conclusions <p>The incidence of anaerobic spondylodiscitis might be underestimated, as it is difficult to recover anaerobic bacteria. Next-generation sequencing demonstrated superior diagnostic accuracy for anaerobic pathogen identification compared to traditional culture methods. Anaerobic spondylodiscitis generally exhibits a favorable prognosis, considering that appropriate antibiotic therapy is administered and suitable surgical treatment is performed if necessary.</p> Clinical trial number <p>Not applicable.</p>