Background: Neonatal sepsis is a clinical emergency that requires sound evaluation supported by accurate laboratory analysis and timely clinical intervention for its management. This study, therefore, was conducted to identify bacteria causing neonatal sepsis and their susceptibility to the commonly prescribed antibiotic at the Neonatal Intensive Care Unit of a tertiary health care facility in the Northern Region of Ghana. Methods: Neonatal biodata were collected from patient folders, after which identification, isolation, and susceptibility of isolated bacteria to prescribed anti-bacterial (Kirby-Bauer disk diffusion method) were carried out on single venipuncture blood samples aseptically drawn from 275 neonates clinically diagnosed with sepsis. Results: 275 neonates took part in the study, of which 218 (79.3%) presented with early-onset sepsis (EOS) and 57 (20.7%) with late-onset sepsis (LOS). The laboratory results confirmed a septicemia prevalence of 70.3% among neonates clinically diagnosed with sepsis. Preterm delivery ( P = .01), hypothermia ( P = .001), and delivery at the tertiary healthcare facility were significantly associated with EOS ( P < .000), while low birth weight ( P = .012), duration of hospital stay ( P = .001), and delivery at the tertiary healthcare facility ( P < .000) were found to be significantly associated with LOS. Gram-positive cocci constituted 54.9% (107), with Gram-negative constituting 45.1% (88) of all the bacteria isolates. Coagulase-negative staphylococcus (CoNS) 70.1% (75) and Klebsiella species 39.8% (35) were the dominant Gram-positive and Gram-negative isolates, respectively. 57.8% and 55.8% of CoNS isolates were susceptible to ampicillin and amoxicillin/clavulanic acid, respectively. 93.5% of CoNS and all the isolated Staphylococcus aureus and Klebsiella species were susceptible to amikacin. Conclusions: Coagulase-negative staphylococcus (CoNS) and Klebsiella species were the predominant Gram-positive and negative sepsis-causing agents at the NICU, respectively. Amikacin exhibited the highest sensitivity to Gram-positive and negative causative agents, making it a strong candidate for consideration in the facility’s empirical treatment of neonatal sepsis.