Background: Seventy–six newborn admitted at NICU were selected to compare the BACTEC 9050 blood culture system and the conventional method. Methods: This comparative study was conducted at the department of microbiology, NIMS, Jaipur from November 2018 to October 2019, and comprised blood samples of suspected newborns. The blood samples were inoculated into automated BACTEC 9050 culture bottles. At the same time, a conventional blood culture bottle was also inoculated for comparison. The antibiotics used for susceptibility testing were penicillin (P), Linezolid, Vancomycin, Ampicillin, Azithromycin, Ceftazidime, Amikacin, Gentamycin, Tetracycline, Clindamycin, Ciprofloxacin, Teicoplanin, Cotrimoxazole, Meropenem, Imipenem, Piperacillin–Tazobactam, Levofloxacin, Cefoxitin, High–Level Gentamycin, Tobramycin. Result: Out of 76 blood culture samples, 52 (68.42%) were positive on BACTEC 9050, while 47 (61.84%) were positive through the conventional method. Out of 47 positive samples, the isolated organisms were Escherichia coli (09), Skin commensal (10) followed by Staphylococcus aureus (11), Klebsiella pneumonia (07), Streptococcus (02), Enterococcus (04), Acinetobacter (2) and Pseudomonas aeruginosa (02). Conclusion: Diagnosis of newborn septicemia through BACTEC 9050 was quicker with high sensitivity compared to the conventional method.