Background In intensive care unit (ICU), infection and colonization by resistant Gram-negative bacteria increase costs, length of stay and mortality. Extended-spectrum beta-lactamase − producing Enterobacteriaceae (ESBL-E) is a group of pathogens increasingly encountered in ICU setting. Conditions that promote ESBL-E acquisition are not completely understood. The increasing incidence of infections related to ESBL-E and the unsolved issues related to ESBL-E cross-transmission, prompted us to assess the rates of referred and acquired cases of ESBL-E in ICU and to assess patient-to-patient cross-transmission of ESBL-E using a multimodal microbiological analysis. Methods During a 5-month period, all patients admitted to a medical ICU were tested for ESBL-E carriage. A rectal swab was performed at admission and then twice a week until discharge or death. ESBL-E strains were analyzed according to antibiotic susceptibility pattern, rep-PCR (repetitive-element Polymerase chain reaction) chromosomal analysis, and plasmid PCR (Polymerase chain reaction) analysis of ESBL genes. Patient-to-patient transmission was deemed likely when 2 identical strains were found in 2 patients hospitalized simultaneously in the ICU. Results Among the 309 patients assessed for ESBL-E carriage on admission, 25 were found to carry ESBL-E (importation rate: 8 %). During follow-up, acquisition was observed among 19 of them (acquisition rate: 6.5 %). Using the multimodal microbiological approach, we found only one case of likely patient-to-patient ESBL-E transmission. Conclusions In unselected ICU patients, we found rather low rates of ESBL-E referred and acquired cases. Only 5 % of acquisitions appeared to be related to patient-to-patient transmission. These data highlight the importance of jointly analyzing phenotypic profile and molecular data to discriminate strains of ESBL-E.