The aim of this study was to identify the bacteria causing neonatal septicemia in a neonatal intensive care unit (NICU) in León, Nicaragua and its relation with bacteria isolated from the environment at the NICU. Our data showed that 74% (34/46) of the bacteria related to newborns with septicemia were Gram-negative and highly resistant to beta-lactams (>85%) and aminoglycosides (80%), leading to treatment failure in 10 neonates with fatal outcome. Although, the prevalence of Gram-positive bacteria (26%) was lower than Gram-negative bacteria, Staphylococcus epidermidis was related to the death of three newborns. No clonal similarity was found among Enterobacter cloacae , Escherichia coli and Serratia liquefaciens isolated from the neonates with septicemia and the NICU environment. However, in order to improve the outcome for neonates with septicemia, infection control practices and appropriate empirical therapy should be considered to reduce the high prevalence of extended-spectrum beta-lactamase (ESBL)-producing Gram-negative bacteria isolated from neonates with septicemia (80%) and from the NICU environment (34%).