Introduction: Infections are a major cause of both morbidity and mortality worldwide. Neonates are vulnerable to a wide range of microorganisms, leading to infections that may be either superficial or systemic. Infants in the NICU face a heightened risk of infections due to several contributing factors. Unlike older children and adults, neonates-especially those born prematurely-have an underdeveloped immune system, making them more susceptible to infections. Objective: To study the microbial profile and determine antibiotic resistance pattern of infections in Neonatal Intensive Care Unit Methodology: This study was a cross-sectional [observational] investigational study conducted in the Central Microbiology Laboratory of Dhiraj General Hospital, SBKS MI & RC DGH & affiliated to Sumandeep Deemed University, Piparia, Waghodia, Gujarat over 1 year approximately from September 2023-September 2024 with a total of 89 culture positive samples of NICU. All the samples that came for the request to carry out culture and AST from NICU. Samples were aseptically collected from NICU including blood, urine, pus, sputum, CSF, endotracheal tube, and others, and were processed further for culture and antibiotic sensitivity testing using VITEK 2. Results: The prevalence of culture-proven septicemia, pneumonia, and meningitis was 5.55% [62 out of 1117], 2.06% [23 out of 1117], and 0.36% [4 out of 1117], respectively in NICU. In the study, of 62 neonatal septicemia cases, 69.35% were early onset and 30.65% were late onset. The study assessed antibiotic sensitivity in neonatal septicemia cases. K. pneumoniae was most sensitive to Cotrimoxazole and A. baumannii to Ceftriaxone. P. aeruginosa and E. coli both showed 100% sensitivity to Cotrimoxazole. MRSA was 100% sensitive to Vancomycin and Linezolid. Conclusion: The study underscores the vital importance of continuous surveillance and antibiotic stewardship in NICU. Given that these units care for highly vulnerable patients, it is essential to consistently monitor infection trends and antibiotic resistance patterns. Regular surveillance enables healthcare professionals to promptly detect emerging threats and adapt treatment protocols to manage these challenges effectively.