Introduction: Given their ability for colonizing the supraglottic region, desiccation tolerance, resistance to β-lactam antibiotics, and adherence to both inert surfaces and epithelial cells, Klebsiella pneumoniae and Escherichia coli are potentially pathogenic microorganisms for patients undergoing mechanical ventilation in an intensive care unit (ICU).Objective: To perform a molecular characterization and detection of extended spectrum β-lactamases (ESBL) in K. pneumoniae and E. coli strains isolated from the supraglottic region of patients undergoing mechanical ventilation in an ICU.Materials and methods: A descriptive study was conducted in 18 isolates. Disk diffusion technique was used for detecting ESBL-producing bacteria. Molecular characterization was made by BOX-PCR technique, while ESBL production was confirmed by testing the isolates against cefotaxime and ceftazidime, alone and in combination with clavulanic acid.Results: a K. pneumoniae strain and another E. coli strain were confirmed as ESBL producers. A divergence greater than 50% was observed in most of the strains; besides non-infectious origin strains resistant to third generation cephalosporins were found.Conclusion: The polyclonality found in this study might indicate that most of the strains belong to each patient’s microbiota.