Objective To investigate the clinical characteristics and impact factors for prognosis of patents with multi-bacteria and mono-bacteria blood stream infection (BSI). Methods Medical records of patients with clinical and pathogenic diagnosis of BSI in a tertiary first-class hospital from January 2017 to December 2021 were retrospectively analyzed.According to the detected species of pathogenic bacteria, patients were divided into multi-bacteria and mono-bacteria groups, the study outcome was all-cause mortality during hospitalization.Clinical characteristics of two groups of patients and impact factors for prognosis of patients with multi-bacteria infection was compared. Results 430 patients were positive in blood culture, 367 cases (85.3%) were infected by mono-bacteria and 63 cases (14.7%) were infected by multi-bacteria.Healthcare-associated infection (HAI) rate in multi-bacteria group was higher than that in mono-bacteria group (76.2% vs 56.9%, P=0.003);lower respiratory tract infection rate in patients in multi-bacteria group was higher than that in mono-bacteria group (44.4% vs 29.2%, P=0.018).Hospitalization time of patients in multi-bacteria group was higher than that in mono-bacteria group (24[16-39]vs 19[13-26]day, P=0.002).Death rate of patients in multi-bacteria group was higher than that in mono-bacteria group (12.7% vs 3.8%), and hospitalization time was prolonged, difference was both significant (both P < 0.05).Multivariate analysis showed that independent risk factors for death in patients with BSI were multi-bacteria infection (OR=3.24, 95%CI=1.20-8.75), gastrointestinal tumor (OR=3.28, 95%CI=1.21-8.84), invasive mechanical ventilation (OR=3.40, 95%CI=1.22-9.42), deep vein catheterization (OR=2.76, 95%CI=1.00-7.64), and urinaryt ract catheterization (OR=3.28, 95%CI=1.04-10.40).Compared with the mono-bacteria group, the interval between specimen submission for detection and patients'discharge in multi-bacteria group was significantly longer (20[15-36]vs 16[10-22]days, P < 0.001).The constituent of Staphylococcus aureus, Staphylococcus haemolyticus and Escherichia coli detected in patients in multi-bacteria group and mono-bacteria group was significantly different (all P < 0.05). Conclusion The risk for death in hospitalized patients with multi-bacteria BSI is significantly increased, digestive tract tumor, invasive mechanical ventilation, deep vein catheterization, and urinary catheterization are independent risk factors for death of patients with BSI, which should be paid attention in clinic.