Abstract Background Multidrug-resistant (MDR) infections are a serious concern for children admitted to the Paediatric Intensive Care Unit (PICU). Tracheal colonization with MDR Enterobacteriaceae predisposes to respiratory infection, but underlying risk factors are poorly understood. This study aims to determine the incidence of children with suspected infection during mechanical ventilation and analyses risk factors for the finding of MDR Enterobacteriaceae in tracheal aspirates. Methods A retrospective single-centre analysis of Enterobacteriaceae isolates from the lower respiratory tract of ventilated PICU patients from 2005 to 2014 was performed. Resistance status was determined and clinical records were reviewed for potential risk factors. A classification and regression tree (CRT) to predict risk factors for infection with MDR Enterobacteriaceae was employed. The model was validated by simple and multivariable logistic regression. Results One hundred sixty-seven Enterobacteriaceae isolates in 123 children were identified. The most frequent isolates were Enterobacter spp., Klebsiella spp. and E.coli. Among these, 116 (69%) isolates were susceptible and 51 (31%) were MDR. In the CRT analysis, antibiotic exposure for ≥ 7 days and presence of gastrointestinal comorbidity were the most relevant predictors for an MDR isolate. Antibiotic exposure for ≥ 7 days was confirmed as a significant risk factor for infection with MDR Enterobacteriaceae by a multivariable logistic regression model. Conclusions This study shows that critically-ill children with tracheal Enterobacteriaceae infection are at risk of carrying MDR isolates. Prior use of antibiotics for ≥ 7 days significantly increased the risk of finding MDR organisms in ventilated PICU patients with suspected infection. Our results imply that early identification of patients at risk, rapid microbiological diagnostics and tailored antibiotic therapy are essential to improve management of critically ill children infected with Enterobacteriaceae.