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Background: Although Staphylococcus aureus bloodstream infections (SA-BSI) are a common and important infection, polymicrobial SA-BSI are infrequently reported. The aim of this study was to investigate the clinical characteristics and risk factors of polymicrobial SA-BSI in comparison with monomicrobial SA-BSI., Methods: A single-center retrospective observational study was performed between Jan 1, 2013, and Dec 31, 2018 at a tertiary hospital. All patients with SA-BSI were enrolled, and their clinical data were gathered by reviewing electronic medical records., Results: A total of 349 patients with SA-BSI were enrolled including 54 cases (15.5%) with polymicrobial SA-BSI. In multivariable analysis, burn injury (adjusted odds ratio [OR], 7.04; 95% confidence interval [CI], 1.71-28.94), need of blood transfusion (aOR, 2.72; 95% CI, 1.14-6.50), use of mechanical ventilation (aOR, 3.11; 95% CI, 1.16-8.30), the length of prior hospital stay (aOR, 1.02; 95% CI, 1.00-1.03), and pneumonia as primary site of infection (aOR, 4.22; 95% CI, 1.69-10.51) were independent factors of polymicrobial SA-BSI. In comparison with monomicrobial SA-BSI, patients with polymicrobial SA-BSI had longer length of ICU stay [median days, 23(6.25,49.25) vs. 0(0,12), p < 0.01] and hospital stay [median days, 50(21.75,85.75) vs. 28(15,49), p < 0.01], and showed a higher 28-day mortality (29.6% vs. 15.3%, p = 0.01)., Conclusions: Burn injury, blood transfusion, mechanical ventilation, the length of prior hospital stay, and pneumonia as a primary site of infection are independent risk factors for polymicrobial SA-BSI. In addition, patients with polymicrobial SA-BSI might have worse outcomes compared with monomicrobial SA-BSI.