Out of the 120 blood samples collected from investigated neonates, 40 samples were positive for bacteria. Blood culture was carried out using BACT/ ALERT (3D 60) then the positive cultures were subculture on MacConkey, Blood, and Sabouraud dextrose agar. Biochemical tests were used to identify the isolates, and the automated Vitek 2 system provided confirmation. The incidence of neonatal sepsis was predominantly due to gram-negative bacteria (75%) mainly K. pneumoniae (60 %), followed that E. coli (7.5 %). While all gram-positive bacteria (25 %) are represented by CONS. As stated by the Clinical and Laboratory Standards Institute (CLSI) guidance, an antimicrobial susceptibility test using the disc diffusion method (Kirby-Bauer) was conducted to select multi-drug resistant bacteria as (ESβL) producers. The ESβL genes responsible for the resistance were detected using the PCR method. It was determined that among ESβL of K. pneumoniae and E. coli, the blaTEM and blaCTX-M genes were the predominant gene (100 %), followed by blaSHV (80 %).