INTRODUCTION: In cases of infectious spondylodiscitis (ISD) where blood cultures are negative, disco-vertebral puncture-biopsy (DVPB) is recommended. In the event of a sterile result, existing literature does not definitively answer the question of whether to initiate empirical antibiotic therapy or to conduct a second DVPB. The aim of this study was to assess the culture yield of DVPB in ISD and to identify the factors associated with a positive DVPB. MATERIALS AND METHODS: A retrospective single-center study was conducted, encompassing all adult patients with ISD having undergone DVPB between 01/01/2009 and 31/10/2021. RESULTS: A total of 177 patients were included. The yield of the first DVPB was 48.6 %. The second DVPB yielded 8.7 % (p <
0.001). Factors significantly associated with the yield of the first DVPB included younger age (p = 0.003), higher CRP levels (p = 0.0496), larger needle size (p = 0.023), and histopathology supporting ISD (p = 0.001), while prior antibiotic therapy (p = 0.001) is a factor associated with negative culture. The second DVPB increased the culture yield by 19.3 %. CONCLUSION: The yield of the second biopsy is lower than that of the first biopsy but provides an additional diagnostic gain of 19.3%. Antibiotic therapy prior to DVB significantly decreases their yield. The utility of routine post-DVPB blood cultures appears to be limited.