The importance of microbial colonization during extracorporeal membrane oxygenation (ECMO) is unclear. We prospectively cultured ECMO oxygenators and retrospectively reviewed the culture results, clinical outcomes, and associated factors in 112 ECMO patients (122 oxygenators, 1,196 ECMO days). Of the oxygenators, 11.6% (n = 13) had positive cultures. The most common pathogen was Klebsiella pneumoniae, followed by Acinetobacter baumannii and Staphylococcus epidermidis. Nine (69%) cases showed catheter colonization, and five (38%) bloodstream infection (BSI) with the same microorganism. Most of the microorganisms were multidrug resistant. BSI tended to be associated with oxygenator colonization (r = 0.172, p = 0.070). The patients were divided into oxygenator colonization (n = 13) and no colonization groups (n = 99). Successful weaning from ECMO and survival to discharge were significantly lower in the oxygenator colonization group (weaning 30.8% vs. 90.9%, p < 0.001, survival 23.1% vs. 76.8%; p < 0.001). In multivariate analyses, age (per decade) (odds ratio [OR] = 1.95, 95% CI = 1.28-2.95; p = 0.002), oxygenator colonization (OR = 15.49, 95% CI = 3.31-72.46; p < 0.001), and renal replacement therapy (OR = 4.61, 95% CI = 1.69-12.58; p = 0.003) were significantly associated with mortality. Oxygenator colonization was associated with poor outcomes in ECMO patients. These results support the early exchange and culture of oxygenators in patients with persisting bacteremia.