Healthcare-associated infections due to multidrug-resistant Gram-negative bacteria (MDR-GNB) are a leading cause of morbidity and mortality worldwide. These evidence-based guidelines have been produced after a systematic review of published studies on infection prevention and control interventions aimed at reducing the transmission of MDR-GNB. The recommendations are stratified by type of infection prevention and control intervention and species of MDR-GNB and are presented in the form of ‘basic’ practices, recommended for all acute care facilities, and ‘additional special approaches’ to be considered when there is still clinical and/or epidemiological and/or molecular evidence of ongoing transmission, despite the application of the basic measures. The level of evidence for and strength of each recommendation, were defined according to the GRADE approach.