Background. Biopsy specimens are often obtained in the evaluation of hematogenous vertebral osteomyelitis. The effect of prebiopsy antibiotic exposure on pathogen recovery is unknown. Methods. We conducted a retrospective cohort study of adult inpatients with hematogenous vertebral osteomyelitis at a tertiary care hospital from 1 January 2003 through 31 July 2007. Antibiotic exposure within 14 days before biopsy was evaluated. Results. Of 150 patients with hematogenous vertebral osteomyelitis, 92 (61%) underwent a biopsy (60 [65%] needle and 32 [35%] open biopsies). The median time from admission to biopsy was 3 days (range, 0-69 days). Patients who underwent biopsy were more likely to have weakness (53 [58%] biopsy vs 15 [26%] no biopsy; P <. 001) and sensory loss (27 [29%] vs 6 [10%]; P =. 006), but were less likely to have a positive blood culture result (28 [30%] vs 30 [52%]; P =. 01). Pathogens were recovered in 61 patients (66%). Open biopsy had a higher yield than needle biopsy (29 [91%] of 32 vs 32 [53%] of 60; P <. 001). Sixty patients (65%) who had biopsies performed received antibiotics ≤14 days before the procedure (median duration, 4 days; range, 1-37 days). Open biopsy predicted positive biopsy culture results (adjusted odds ratio, 8.4; 95% confidence interval, 2.2-31.8), but there was no association of prebiopsy antibiotics with culture results (adjusted odds ratio, 2.3; 95% confidence interval, 0.8-6.2). Conclusions. A pathogen was recovered from 61 (66%) of 92 patients who had biopsies performed in this cohort of hematogenous vertebral osteomyelitis. Open biopsies had a higher microbiological yield than did needle biopsies. Antibiotic exposure before biopsy did not negatively impact pathogen recovery and should not be the sole reason for foregoing biopsies.