【Background】 Identification of pathogenic microorganisms are essential for pyogenic vertebral osteomyelitis (PVO). This study aimed to demonstrate the effectiveness of the blood culture bottle (BCB) system in identifying PVO causative organisms. 【Materials & Methods】 We analyzed retrospectively collected data from patients who underwent full-endoscopic spine surgery for PVO between January 2016 and March 2019. Irrigation water generated in the surgical field was incubated in the BCB system, and compared with blood culture before surgery and tissue culture taken by the conventional method. The microbial identification rate and the time from sample collection to microbial identification of the BCB system were compared with conventional blood culture and tissue cultures using the Wilcoxon signed-rank test. 【Results】 We included 17 patients (12 men, five women; mean age, 72.8 ± 11.9 years). Bacteria were cultured from 3 (17.6%), 13 (76.5%), and 12 (70.6%) patients by blood culture, tissue culture, and the BCB system, respectively. Tissue culture and the BCB system had significantly higher detection rates than blood culture (P = .002, P =.003), and there was no significant difference between tissue culture and BCB system (P = .655). In 15 cases (88.2%), the causative organism was identified by at least one method. In two cases, the causative organism (Escherichia coli) was only identified by the BCB system. The BCB system required amount of time for microbial identification (3.9 ± 3.0 days), compared with the time required for blood culture (5.0 ± 1.4 days, P =.180) and tissue culture (11.9 ± 15.1 days, P =.012). 【Conclusions】 Results suggest the possibility of improving the detection rate and time to detection of causative organisms by the BCB system as an adjunct to the conventional method.