Background: Cross-transmission of nosocomial pathogens occurs frequently in intensive care units (ICU). The aim of this study was to investigate whether the introduction of a single room policy resulted in a decrease in transmission of multidrug-resistant (MDR) bacteria in an ICU. Methods: We performed a retrospective study covering two periods: between January 2002 and April 2009 (old-ICU) and between May 2009 and March 2013 (new-ICU, single-room). These periods were compared with respect to the occurrence of representative MDR Gram-negative bacteria. Routine microbiological screening, was performed on all patients on admission to the ICU and then twice a week. Multi-drug resistance was defined according to a national guideline. The first isolates per patient that met the MDR-criteria, detected during the ICU admission were included in the analysis. To investigate the clonality, isolates were genotyped by DiversiLab (bioMérieux, France) or Amplified Fragment Length Polymorphism (AFLP). To guarantee the comparability of the two periods, the 'before' and 'after' periods were chosen such that they were approximately identical with respect to the following factors: number of admissions, number of beds, bed occupancy rate, per year and month. Results: Despite infection prevention efforts, high prevalence of MRD bacteria continue to occur in the original facility. A marked and sustained decrease in the prevalence of MDR-GN bacteria was observed after the migration to the new ICU, while there appear to be no significant changes in the other variables including bed occupancy and numbers of patient admissions. Conclusion: Single room ICU design contributes significantly to the reduction of cross transmission of MRD-bacteria.