Background <p>Microbiological diagnosis is crucial for managing vertebral osteomyelitis (VO), but the impact of pre-culture antimicrobial therapy on culture positivity remains debated. This study aimed to investigate the association between pre-culture antimicrobial administration and the positivity of comprehensive culture tests in patients with VO.</p> Methods <p>This single-center cohort study included adult patients who were diagnosed with VO and admitted to a university hospital in Japan between 2006 and 2021. The primary outcome was the proportion of positive culture tests (blood, bone, or abscess). Pre-culture antimicrobial therapy was defined as receiving at least one systemic dose within two days before the first culture specimen collection. To adjust for confounding factors, we used the inverse probability of treatment weighting to estimate average treatment effects (ATEs) and 95% confidence intervals (CIs).</p> Results <p>Of the 133 patients, 46 (34.6%) received pre-culture antimicrobials. Overall, 65.4% of patients had at least one positive culture. Pre-culture antimicrobial therapy was significantly associated with a lower proportion of any positive culture (ATE − 0.22; 95% CI, -0.39 to -0.05). When analyzed by specimen type, significant negative associations were found for blood cultures (ATE − 0.30; 95% CI, -0.48 to -0.12) and abscess cultures (ATE − 0.33; 95% CI, -0.51 to -0.14). However, no significant association was observed for bone cultures (ATE − 0.07; 95% CI, -0.33 to 0.20). The duration of antimicrobial therapy before culture collection was not significantly associated with culture positivity.</p> Conclusion <p>Pre-culture antimicrobial therapy was associated with a 20% to 30% lower absolute proportion of positive blood and abscess cultures but did not significantly affect the positivity of bone cultures in patients with VO. Bone biopsies may remain a valuable diagnostic tool even if antimicrobial agents have been administered.</p>