In developing nations, neonatal sepsis is still a major leading health concern, especially with the increasing threat posed by antimicrobial resistance (AMR) and the prevalence of multidrug-resistant (MDR) organisms. The objective of this study is to examine the resistance patterns of antibiotics and assess the occurrence of multidrug-resistant organisms in neonatal sepsis cases at a tertiary care hospital in India. A cross-sectional observational study was conducted from July 2023 to February 2025 in the NICU of a tertiary care hospital. We included all neonates after 35 weeks of gestation with suspected sepsis admitted to the Department of Pediatrics, SGT Hospital. Neonates who had received antibiotics for more than 24 hours prior to admission were excluded. The sample size was calculated using the single population proportion formula, considering the prevalence of neonatal sepsis to be 20.7%. The calculated sample size was 253; however, we planned to enroll 300 neonates to obtain a round figure and ensure adequate representation. Blood specimens from 300 neonates presenting with clinical signs of sepsis were processed for culture using the automated BACTEC system. Bacterial isolates were characterized, and their antibiotic resistance profiles were determined following established standard procedures. Data were analysed using Microsoft Excel. Of 300 neonates, 68 (23%) had culture-confirmed sepsis. Gram-positive cocci (66.18%) were more prevalent than Gram-negative bacilli (33.82%). The predominant isolates included Coagulase-negative Staphylococci (61.36%), Klebsiella pneumoniae (34.78%), and Acinetobacter spp. (21.74%). Gram-positive isolates showed high resistance to erythromycin (44.44%) and penicillin (38.88%), while Gram-negative isolates demonstrated the highest resistance to ciprofloxacin, levofloxacin, piperacillin-tazobactam, and gentamicin (47.82% each). Multidrug-resistance was common, notably in Klebsiella pneumoniae (87.5%), Acinetobacter lwoffii (50%), Escherichia coli (50%), Staphylococcus saprophyticus (100%), and Streptococcus mitis (100%). The high prevalence of multidrug-resistant organisms, especially among Gram-negative isolates, highlights an urgent need for strengthened antimicrobial stewardship through measures such as rational antibiotic use, periodic antibiotic susceptibility surveillance, and implementation of strict infection control practices including hand hygiene, aseptic procedures, and isolation of resistant cases. Continuous surveillance and rational antibiotic use are essential to mitigate the threat of AMR in neonatal care settings.