To explore the influencing factors of spinal mixed infection under mNGS-assisted detection. A retrospective analysis was conducted on the general clinical data of patients diagnosed with spinal infections at Guilin People's Hospital, covering the period from October 2022 to October 2024, to evaluate the effectiveness of different treatment modalities including conservative, pharmacological, and surgical interventions. In the end, a total of 45 cases were included, including 18 cases of mixed infection and 27 cases of single infection. The receiver operating characteristic (ROC) curve was utilized to evaluate the predictive efficacy of various indices for the occurrence of mixed infection in patients with spinal infections, with the curve's proximity to the top left corner indicating higher diagnostic accuracy. Multivariate Logistic regression was used to analyze the independent risk factors affecting the development of mixed infection in patients with spinal infection. No significant differences were found between the two groups regarding gender, smoking, alcohol consumption, hypertension, albumin levels, liver function, malignancy, or rheumatic immune disease history (P > 0.05). However, the mixed infection group had significantly higher proportions of patients aged > 60 years (78% (14/18) vs. 48% (13/27)), diabetes mellitus (44% (8/18) vs. 15% (4/27)), chronic kidney disease (17% (3/18) vs. 0.00 (0/27)), and previous spinal surgery (39% (7/18) vs. 11% (3/27)), along with lower BMI (20.70 ± 2.15 vs. 24.04 ± 3.76) and hemoglobin levels (105.17 ± 14.05 g/L vs. 117.48 ± 18.08 g/L). The results of the ROC curve analysis showed that the area under the curve for BMI and hemoglobin in predicting the occurrence of mixed infection in patients was 0.787 and 0.704, respectively, with optimal cutoff values of 21.12 kg/m and 119 g/L. Multivariate logistic regression identified BMI < 21.12 kg/m, hemoglobin < 119 g/L, and diabetes as independent risk factors. Lower BMI, diabetes and hemoglobin are independent risk factors for spinal mixed infection. Increasing BMI, effectively controlling blood glucose and improving anemia are helpful to reduce the occurrence of spinal mixed infection.