Background: This study aimed to determine the epidemiology of gram-negative bacteria (GNB) isolated in the newborn intensive care unit (NICU) population, to assess their antibiotic susceptibility patterns and possible associated risk factors.Methods: All neonates admitted to the NICU of ABDERREZAK-BOUHARA hospital (Skikda, Algeria) with a clinical diagnosis of neonatal infections from March to May 2019 were included in the study. The extended-spectrum beta-lactamase (ESBLs), plasmidic cephalosporinase (pAmpC), and carbapenemases genes were screened by polymerase chain reaction (PCR) and sequencing. PCR amplification of oprD among carbapenem-resistant Pseudomonas aeruginosa isolates was also performed. The clonal relatedness of the ESBLs isolates was studied using multilocus sequence typing (MLST).Results: Among 148 clinical specimens, 36 (24.3%) GNB strains were isolated from urine (n = 22), wound (n = 8), stool (n = 3), and blood (n = 3) samples. The bacterial species identified were Escherichia coli (n = 13), Klebsiella pneumoniae (n = 5), Enterobacter cloacae (n = 3), Serratia marcescens (n = 3), Salmonella spp. (n = 3), Proteus mirabilis (n = 1), P. aeruginosa (n = 5), and Acinetobacter baumannii (n = 3). PCR and sequencing showed that eleven Enterobacterales isolates harbored the bla(CTX-M-15) gene, two E. coli isolates harbored the bla(CMY-2) gene, and three A. baumannii isolates harbored both bla(OXA-23) and bla(OXA-51) genes. Also, five strains of P. aeruginosa were found to harbor mutations in the oprD gene. MLST showed that the K. pneumoniae strains belonged to ST13 and ST189, E. coli belonged to ST69, and E. cloacae belonged to ST214. Different risk factors that could predict positive GNB cultures were found, including female sex, Apgar score <8 at 5 min of life, enteral nutrition, antibiotic use, and extended length of hospitalization.Conclusion: Our study highlights the importance of determining the epidemiology of pathogens causing neonatal infections, their sequence types (ST), and their antibiotic susceptibility patterns to address rapidly a correct antibiotic treatment regimen.