We performed a prospective study to evaluate the clinical usefulness of a direct 16S rRNA gene (16S rDNA) PCR assay of percutaneous biopsies or aspirates for the etiological diagnosis of vertebral osteomyelitis. During May 2009 to December 2010 and November 2011 to August 2012, consecutive patients with suspected vertebral osteomyelitis who underwent a percutaneous biopsy or aspiration were enrolled. Of 45 patients with vertebral osteomyelitis, 16S rDNA PCR was positive in 24 (53.3%), whereas culture was positive in 13 (28.9%) (P = 0.027). Three of PCR-positive cases (12.5%, 3/24) and 1 of culture-positive case (7.7%, 1/13) were considered to be false-positives. Of 16 patients without prior antimicrobial exposure, 75% of cases (12/16) were positive by either culture (7/16, 43.8%) or PCR (9/16, 56.3%). A 16S rDNA PCR assay with sequencing was more sensitive than routine culture for the etiological diagnosis of vertebral osteomyelitis.