Health care associated infections (HAIs) are currently among the major challenges to the care of hematopoietic stem cell transplantation (HSCT) patients. The objective of the present study was to evaluate the impact of 2% chlorhexidine (CHG) bathing on the incidence of colonization and infection with vancomycin-resistant Enterococcus (VRE), multidrug-resistant (MDR) gram-negative pathogens, and to evaluate their CHG minimum inhibitory concentration (MIC) after the intervention.A quasi-experimental study with duration of 9 years was conducted. VRE colonization and infection, HAI rates, and MDR gram-negative infection were evaluated by interrupted time series analysis. The antibacterial susceptibility profile and mechanism of resistance to CHG were analyzed in both periods by the agar dilution method in the presence or absence of the efflux pump inhibitor carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and presence of efflux pumps (qacA/E, qacA, qacE, cepA, AdeA, AdeB, and AdeC) by polymerase chain reaction (PCR).The VRE colonization and infection rates were significantly reduced in the postintervention period (P = 0.001). However, gram-negative MDR rates in the unit increased in the last years of the study. The CHG MICs for VRE increased during the period of exposure to the antiseptic. A higher MIC at baseline period was observed in MDR gram-negative strains. The emergence of a monoclonal Pseudomonas aeruginosa clone was observed in the second period.Concluding, CHG bathing was efficient regarding VRE colonization and infection, whereas no similar results were found with MDR gram-negative bacteria.