Infection is one of the most frequent complications in the management of ECMO patients. Although there are several studies demonstrating the role of ECMO in patients with pre-existing sepsis, there are only a few studies with relatively small size on new-onset blood stream infection (BSI) during ECMO. Our study aimed to determine the clinical impact of new-onset BSI in ECMO. This was a retrospective, single-center and observational study. A total of 852 consecutive patients underwent ECMO at our institution between January 2014 and June 2020. Excluding patients with documented infections before ECMO, 639 patients were enrolled for analysis. In the multivariable analysis, ECMO weaning failure was associated with BSI (P = 0.005), continuous renal replacement therapy (P = 0.001), and lactate level at 24 h after ECMO (P = 0.004). Overall mortality was independently associated with BSI (P = 0.008). BSI was identified in 110 patients (17.2%), and these patients experienced more concomitant infections (78.2% vs. 38.8%; P < 0.001), red blood cell transfusions (17.0 [10.0, 30.0] vs. 9.0 [4.0, 17.0] units; P < 0.001), limb ischemia (12.7% vs. 5.3%; P = 0.008) and gastrointestinal bleeding (17.3% vs. 3.4%; P < 0.001) than patients without BSI. In conclusion, newly developed BSI during ECMO is significantly associated with poor clinical outcome. Pre-emptive management of patients with high risk of BSI may improve both ECMO weaning success rate and overall survival.