The aims of this study are to assess the diagnostic yield of image-guided biopsy for discitis-osteomyelitis (DO), identify factors associated with biopsy yield (laboratory, pre-defined MRI findings, and biopsy technique), and impact of biopsy on management of patients appropriately selected according to the Infectious Disease Society of America guidelines (IDSA). This is a retrospective review of patients who underwent biopsy for suspected DO from 2011 to 2019. Reference standards to establish diagnosis of DO in order were histopathology/microbiology from biopsy or subsequent surgical sampling, positive blood culture or serology, and imaging/clinical follow-up. Laboratory markers, pre-biopsy antibiotics and MRI features, procedural-related variables, and impact of biopsy on management were assessed. Multivariable logistic regression was also performed. Out of 97 included patients, 78 were diagnosed with DO. Overall sensitivity of biopsy for detecting DO was 41.0% (32/78), including 10 patients with positive histopathology only, 14 with positive biopsy culture only, and 8 with both. Elevated ESR (p < 0.001) and epidural collection on MRI (p = 0.008) were associated with higher biopsy yield (63.6% and 68.6%, respectively) in a multivariable model. Procedural variables were not associated with yield. Biopsy results impacted the management in 19/77 (24.7%) patients, of whom 15/19 (78.9%) had treatment de-escalation and 4/19 (21.0%) had treatment escalation including starting new anti-tuberculous and anti-fungal regimens. Sensitivity of biopsy for detecting DO was 41.0%. When IDSA guidelines are followed, biopsy provided impactful information that changed the management in 24.7% of patients. Evaluation for elevated ESR and epidural collection can help improve yield and patient selection for biopsy.