Identifying the causative pathogens in patients with pyogenic vertebral osteomyelitis is important for optimizing antibiotic therapy. Thus, we examined patient characteristics and investigated the efficacy of the combined use of blood culture and image-guided biopsy culture for identifying the causative pathogens of pyogenic vertebral osteomyelitis. A case series study was conducted in hospitalized patients who were diagnosed with pyogenic vertebral osteomyelitis using electronic medical records. Twenty-three patients who had undergone both blood culture and image-guided biopsy to determine the causative pathogens were identified. Detailed patient characteristics were identified, and the detection rate of a causative pathogen was 57% using either blood or biopsy specimen culture. By combining the results of each culture, the detection rate was increased to 83%. We described the detailed characteristics of patients with pyogenic vertebral osteomyelitis from whom both blood and biopsy specimen cultures were obtained. We found that the combined use of blood culture and biopsy specimen culture is useful for enhancing the detection of causative pathogens in patients with pyogenic vertebral osteomyelitis.