Bloodstream infections (BSIs) are associated with a significantly increased risk of fatality. No report has been found about the molecular epidemiology of Enterobacteriaceae causing BSI in neonates in Poland. The aim of this work was to determine the antibiotic resistance profiles, virulence gene prevalence, the epidemiological and genetic relationships among the isolates from Enterobacteriaceae causing BSI in neonates with birth weight < 1501 g. Antimicrobial susceptibility testing was performed. PCR was performed to identify the presence of common beta-lactamase genes, virulence genes. PFGE and MLST were performed. The surveillance group contained 1,695 newborns. The incidence rate for BSIs was 5.9%, the fatality rate 15%. The most common species were Escherichia coli (n = 24) and Klebsiella pneumoniae (n = 16). CTX-M-15 was found in 6 E. coli, 8 K. pneumoniae, 1 Enterobacter cloacae strains. Among E. coli fimH (83.3%), ibeA (37.5%), neuC (20.8%) were the most frequent. PFGE demonstrated unique pulsotypes among E. coli. E. coli ST131 clone was found in 7 E. coli strains. PFGE of 16 K. pneumoniae strains showed 8 pulsotypes. Five isolates from one NICU belonged to one clone. MLST typing revealed 7 different ST with ST336 as the most prevalent. This study provides information about resistance, virulence and typing of Enterobacteriaceae strains causing BSI among neonates. E. coli and Klebsiella spp. isolated in this study have completely different epidemiology from each other.