AbstractTo assess drug-resistant bacterial colonisation rates and associated risk factors in children with complex chronic conditions admitted to a national reference unit in Spain. Cross-sectional study that included all children admitted to our unit from September 2018 to July 2019. Rectal swabs were obtained to determine multidrug-resistant Gram-negative bacilli (MR-GNB) colonisation, and nasal swab to determine S. aureus and methicillin-resistant S. aureus (MRSA) colonisation. Medical records were reviewed. 100 children were included, with a median of four complex chronic conditions. Sixteen percent had S. aureus colonisation, including two MRSA. S. aureus colonisation was associated with technology-dependent children, while being on antibiotic prophylaxis or having undergone antibiotic therapy in the previous month were protective factors. The prevalence of MR-GNB colonisation was 27%, which was associated with immunosuppressive therapy (aOR 31; 2.02–47]; p = 0.01), antibiotic prophylaxis (aOR 4.56; 1.4–14.86; p = 0.012), previously treated skin-infections (aOR 2.9; 1.07–8.14; p = 0.03), surgery in the previous year (aOR 1.4; 1.06–1.8; p = 0.014), and hospital admission in the previous year (aOR 1.79; [1.26–2.56]; p = 0.001). The rate of S. aureus nasal colonisation in this series was not high despite the presence of chronic conditions, and few cases corresponded to MRSA. Antibiotic prophylaxis, immunosuppressive therapies, history of infections, previous surgeries, and length of admission in the previous year were risk factors for MR-GNB colonisation.