Background: Escherichia coli is one of the most frequent causes of late-onset neonatal sepsis. The aim of this study was to characterize an outbreak of neonatal sepsis occurring in the neonatal intensive care unit (NICU) of the Hospital Clinic of Barcelona from April to August 2013. Methods: After presentation of the index case, all E. coli isolates from previously hospitalized neonates, health care workers, and neonates admitted to the NICU from April to October 2013 were tested for K1 antigen positivity and epidemiologically compared by pulse-field gel electrophoresis. Furthermore, the E. coli K1 strains collected from neonates during this period were analyzed by different methods (serotyping, phylotyping, PCR of virulence factors, antimicrobial resistance, and 'in vitro' assays in HMBEC). Results: An E. coli O18:K1:H7 sequence type 95 and phylogenetical group B2 strain was the cause of the outbreak involving 6 preterm neonates: one with late septicemia due to a urinary focus and 5 with late-onset septicemia and meningitis, 3 of whom died. All showed the same pulsotype, full resistance to ampicillin and intermediate resistance to gentamicin. The outbreak strain carried the PAI IIJ96-like domain that could explain the high-grade bacteremia necessary to develop meningitis. Conclusions: All the E. coli isolates responsible for this outbreak belonged to a single clone suggesting a common source of infection, and it was categorized as O18:K1:H7. Despite the bacteria's pathogenicity has an important role in the severity of infection, the host-associated factors were crucial for the fatal outcomes.