Surveillance strategies to detect colonization is an important tool to prevent and control the spread of microorganisms especially among Hematopoietic Stem Cell Transplant (HSCT) patients. Colonization by Multidrug-resistant organisms (MDRO) has been evaluated as a risk factor for blood stream infection (BSI) in HSCT patients. The aim of this study was to evaluate the use of routine surveillance culture to screening colonization and infection by carbapenem-resistant Enterobacteriaceae (CRE), carbapenem-resistant Pseudomonas aeruginosa (CRPa) and vancomycin-resistant enterococci (VRE) in a HSCT unit. Methods Surveillance cultures were collected from patients admitted to the HSCT unit over one-year, with swabs for cultures on admission and then weekly until discharge. We compared surveillance culture positivity for each site and agent, also clinical and epidemiological data according to the colonization status. Results 200 HSCT patients underwent surveillance, with 1.323 samples collected. Infection due to MDRO occurred in 52 (21.5%) patients, among them 45 (86.5%) were blood stream infection (BSI) and 12 (23%) had positive surveillance culture before infection. 554 (41.8%) surveillance cultures were performed for CRPa, 413 (31.2%) for VRE, and 356 (27%) for CRE. Of these, 179 (13.5%) surveillance culture were positive, with greater positivity for oropharynx (6, 35.3%) CRPa, and rectal samples (16, 20.7%) for CRE. Being colonized by any MDRO, CRE (p <0.001) and CRPa (p = 0.027) was associated with a higher risk of infection in the bivariate analysis but being colonized was not associated with risk of death. Conclusion Previous colonization by MDRO was a significant risk factor for infection by these pathogens, mainly colonization by CRE. Overall, rectal swab was the best site with the higher positivity, and the oropharynx was also an option for CRPa investigation. Feces culture showed low positivity and should be avoided. Although the impact of the strategy on the mortality of patients undergoing HSCT is not clear, VRE surveillance should be questioned in auto-HSCT patients as it has an additional cost and little impact on survival.