Background: Gram-negative bacteria are a primary source of illness in newborns. Objective: The purpose of this study was to isolate, characterise, and analyse the antibiotic resistance patterns of some Gram-negative bacteria that are involved in illnesses in newborns in Ebonyi State. Methods: A total of 200 unique blood and stool samples were gathered from infants in the Nursery Unit of Mile Four Hospital Abakaliki. Demographic information was taken from the patients' medical records. The samples were promptly sent to the Applied Microbiology Laboratory Complex at Ebonyi State University Abakaliki for bacteriological testing. The examination followed conventional microbiological protocols for isolating and identifying bacteria. The isolates underwent antibiotic susceptibility testing using the Kirby – Bauer disc diffusion method. The results were interpreted using the Clinical Laboratory Standard Institute (CLSI) zone diameter breakpoints, and the multiple antibiotic resistance index (MARI) was established. Findings: The findings indicated that there was a total isolation rate of 17.5%, with E. coli and K. pneumoniae isolates accounting for 9.5% and 8% respectively. The demographic data of patients indicated that the isolation rate was 54.5% for male patients and 45.5% for female patients. The K. pneumoniae and E. coli isolates showed a high level of resistance, ranging from 50% to 100%, against amikacin, ertapenem, meropenem, ceftazidime, ceftriaxone, cefepime, sulphamethoxazole, and novobiocin. They also exhibited multi-drug resistance (MDR) with a minimum inhibitory concentration (MIC) value of ≥0.5. However, they were completely susceptible to gentamicin, ofloxacin, and ciprofloxacin. Conclusion: This analysis supports the need of regularly washing hands and using gloves among healthcare personnel and hospital residents. Thoroughly examining pregnant mothers and newborns is crucial in order to minimise the potential hazards linked to probable exposure to K. pneumoniae and E. coli.