Introduction Febrile neutropenia is one of the most serious treatment-related complications in cancer patients. Susceptible to rapidly progressing infections, which result in prolonged hospitalization and use of broad-spectrum antibiotics, neutropenic patients are subject to colonization by multiresistant agents, which enhances the risk of infections.Methods In this study we included samples collected with nasal, oropharyngeal and anal swabs from hospitalized children with febrile neutropenia following chemotherapy, between March 2014 and 2015, aiming to elucidate colonization by S. aureus and Enterococcus spp., as well as their resistance profile.Results S. aureus was found in 22% of the patients and 14% of the events. Methicillin-resistant S. aureus colonized 13.6% of patients. Including anal swabs in the screening increased the identification of colonized patients by 20%. Enterococcus spp. was found in 27% of patients and 17% of episodes.Enterococcal isolates resistant to vancomycin, accounting for 25% of the total, were not isolated in anal swabs at any time, with the oropharyngeal site being much more important. The rate of infection by Enterococcus spp. was 4.5% of all patients and 16% among the colonized patients. ConclusionEspecially in this population, colonization studies including more sites can yield a higher chance of positive results. Establishing the colonization profile in febrile neutropenic children following chemotherapy may help to institute an empirical antibiotic treatment aimed at antibiotic adequacy and lower induction of resistance, thereby decreasing the risk of an unfavorable clinical outcome.