Neonatal septicemia is a major cause of infection and mortality in neonates. Neonatal sepsis is a type of neonatal infection, particularly bacteremia, diagnosed by the presence of microorganisms in blood via microbiological culture tests during the first 4 weeks of birth. Neonatal sepsis is classified into early bacterial neonatal septicemia (less than 72 h) and late bacterial neonatal septicemia (>72 h–28 d) according to age. Isolation of septicemia-causing bacteria from the blood of neonates and investigation of their antibiotic susceptibility pattern for disease management. This study was a retrospective analysis of blood cultures from neonates admitted to the NICU with symptoms of septicemia from June to December, 2019. All samples were processed according to standard microbiological procedures using the BACTEC 9050 automated culture system. Results were reported on the basis of microbial appearance on the culture media and microscopic examination using the Gram staining method, and authentication was performed using standard biochemical reactions. Of the 228 patients, 32.89% were positive for blood culture. Early bacteremia was detected in 64% of the patients, and late bacteremia was detected in 36% of the patients. The most effective drugs against the gram-positive bacteria were linezolid, vancomycin, and ciprofloxacin and against the gram-negative bacteria were tigecycline, imipenem, and amikacin. Coagulase-negative staphylococci and gram-negative organisms such as Klebsiella, Acinetobacter, and Staphylococcus aureus were the major cause of neonatal septicemia in this study, and almost all the isolates were resistant to different antibiotics. Overall, antimicrobial drug resistance was observed in all the positive blood culture samples.