Despite the advantages of extracorporeal membrane oxygenation (ECMO), secondary catheter infection remains a major concern during ECMO support. In this study, to clarify the mechanism of ECMO catheter-related infection, we evaluated the impact of infection on biofilm formation on the surfaces of ECMO catheters, and we investigated clinical factors associated with biofilm formation. Catheters used for ECMO were prospectively collected aseptically from the femoral vein, internal jugular vein, and femoral artery of 81 patients with acute cardiorespiratory failure between January 2015 and October 2016. Prepared catheter sections were investigated by fluorescence microscopy, confocal scanning laser microscopy, transmission electron microscopy, and using semiquantitative culture methods. Of the 81 patients, 51 were assigned to the infection group and 30 to a control group. Biofilms were identified in 43.1% patients in the infection group, and in 20% controls (p = 0.034). Extracorporeal membrane oxygenation flow, systemic infection, and carbapenem-resistant Acinetobacter baumannii (CRAB) infection were associated with biofilm formation in a univariate analysis (odds ratio [OR]: 1.00, 95% confidence interval [CI]: 1.00–1.00, p = 0.007; OR: 3.03, 95% CI: 1.06–8.69, p = 0.039; OR: 9.60, 95% CI: 2.94–31.30, p < 0.001, respectively). However, of these factors, only CRAB infection was found to independently predict the presence of a biofilm by a multivariate logistic regression analysis (OR: 9.60, 95% CI: 2.94–31.30; p < 0.001). Biofilms were more prevalent in patients with an infection than in uninfected controls. Carbapenem-resistant A. baumannii infection was identified as an independent risk factor for biofilm formation on ECMO catheters.