Background and Aims Neonatal infections caused by Extended-spectrum beta-lactamase (ESBL)-producing bacteria are associated with increased morbidity and mortality. No data are available on neonatal colonization with ESBL-producing bacteria in Ecuador. The aim of this study was to determine the proportion of intestinal colonization with ESBL-producing Enterobacteriaceae, their resistance pattern and risk factors of colonization in a neonatal intensive care unit in Ecuador. Methods During a three month period, stool specimens were collected every two weeks from hospitalized neonates. Species identification and susceptibility testing were performed with Vitek2, epidemiologic typing with automated repetitive PCR. Associations between groups were analyzed using the Pearson X2 test and Fisher exact test. A forward step logistic regression model identified significant predictors for colonization. Results Fifty-six percent of the neonates were colonized with ESBL-producing Enterobacteriaceae. Length of stay longer than 20 days and enteral feeding with a combination of breastfeeding and formula feeding were significantly associated with ESBL-colonization. The strains found were E. coli (EC, 89%) and K. pneumoniae (KP, 11%) and epidemiological typing divided these isolates in two major clusters. All EC and KP had blaCTX-M group 1 except for a unique EC isolate that had blaCTX-M group 9. Multi-locus sequence typing performed on the K. pneumoniae strains showed that the strains belonged to ST855 and ST897. The two detected STs belong to two different epidemic clonal complexes (CC), CC11 and CC14, which previously have been associated with dissemination of carbapenemases. None of the E. coli strains belonged to the epidemic ST 131 clone. Conclusions More than half of the neonates were colonized with ESBL-producing Enterobacteriaceae where the main risk factor for colonization was length of hospital stay. Two of the isolated clones were epidemic and known to disseminate carbapenemases. The results underline the necessity for improved surveillance and infection control in this context.Cuencavol. 8; no. 10