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Introduction: Febrile neutropenia (FN) is the most frequent hemato-oncological emergency, with high morbidi ty and mortality in pediatrics. The objective of the study was the microbiological characterization and antimicrobial susceptibility of infections associated with FN in pediatric hemato-oncological patients., Patients and Method: Retrospective cohort study with patients aged between 1 month and 18 years, with onco-hematological pathology according to ICD-10 codes, hospitalized in a tertiary healthcare center in Bucaramanga, Colombia. Based on the medical records of the period 2013-2017, the episodes of FN were identified, and the isolated microorganisms and their susceptibility pattern were described. Biochemical identification and antimicrobial susceptibility testing were performed with the Dade Behring Microscan« automated system. The resistant microorganism classification was performed based on the Minimum Inhibitory Concentration (MIC) and the interpretation of the laboratory according to the cut-off points of the Clinical and Laboratory Standards Institute recommendations., Results: Of 130 patients, 14.7% of the cultures obtained were positive. Bloods tream infection was observed in 17.5% of the episodes. The isolated microorganisms were mainly Gram-negative bacteria (75.8%). Enterobacteriaceae (EB) were the most frequent, led by Klebsiella pneumoniae, Escherichia coli, followed by Pseudomonas aeruginosa and coagulase-negative Staphylo cocci. Of the EBs, 40.5% showed resistance to Piperacillin/Tazobactam, 33.3% to Cefepime, and 8.2% to Meropenem. According to the antimicrobial resistance pattern, it was observed that 16.4% of the positive EB cultures had an extended-spectrum beta-lactamase pattern and 5% a pattern suggestive of carbapenemases. All Gram-positive cocci were sensitive to Vancomycin., Conclusion: In the studied patients, the predominant pathogenic microorganisms were Gram-negative ones with resistance in dices similar to those of developing countries.