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Little is known about cannula-related infection (CRI) in patients supported by extracorporeal membrane oxygenation (ECMO). The aim of this study was to assess the incidence, the risk factors, prognosis, and microbiological characteristics of CRI in patients supported by ECMO. This retrospective cohort study was conducted in one intensive care unit (ICU). Among 220 consecutive patients with peripheral ECMO, 39 (17.7%) developed CRI. The incidence of CRI was 17.2 per 1,000 ECMO days. The main isolated microorganisms were Enterobacteriaceae (38%), Staphylococcus spp. (28.2%; 8.5% were methicillin-sensitive Staphylococcus aureus and 19.7% were coagulase-negative staphylococci), and Pseudomonas aeruginosa (18.3%). Bacteremia was present in 23 cases (59.7%). In multivariate analysis, the risk factors for CRI were longer ECMO duration (p = 0.006) and higher Simplified Acute Physiology Score 2 (p = 0.004). Forty-one percentage of patients with CRI needed surgical management of the infected site. Cannula-related infection was not associated with higher in-hospital mortality (p = 0.73), but it was associated with a longer stay in ICU (p < 0.0001) and a longer stay in hospital (p = 0.002). In conclusion, CRI is frequent in patients with ECMO and associated with a longer stay in hospital. Risk factors for CRI were longer ECMO duration and higher Simplified Acute Physiology Score 2. Concomitant bacteremia was frequent (59.7%) and CRI should be strongly investigated in cases of positive blood culture.