The purpose of this investigation was to evaluate the microbiological diagnosis yield of post-biopsy blood cultures (PBBCs) and second percutaneous needle biopsy (PNB) following an initial negative biopsy in vertebral osteomyelitis (VO) without bacteremia. A retrospective multicenter study was performed. Patients with VO, pre-biopsy negative blood culture(s), ≥1 PNB, and ≥1 PBBC (0-4 h) were included. One hundred and sixty-nine PNBs (136 first and 33 following initial negative biopsy) were performed for 136 patients (median age = 58 years, sex ratio M/F = 1.9). First and second PNBs had a similar yield: 43.4 % (59/136) versus 39.4 % (13/33), respectively. Only two PBBCs (1.1 %) led to a microbiological diagnosis. The strategy with positive first PNB and second PNB following an initial negative result led to microbiological diagnosis in 79.6 % (74/93) of cases versus 44.1 % (60/136) for the strategy with only one biopsy. In the multivariate analysis, young age (odds ratio, OR [95 % confidence interval (CI)] = 0.98 [0.97; 0.99] per 1 year increase, p = 0.02) and >1 sample (OR = 2.4 ([1.3; 4.4], p = 0.007)) were independently associated with positive PNB. To optimize microbiological diagnosis in vertebral osteomyelitis, performing a second PNB (after an initial negative biopsy) could lead to a microbiological diagnosis in nearly 80 % of patients. PBBC appears to be limited in microbiological diagnosis.