BACKGROUND: Early-onset sepsis (EOS) remains a significant cause of morbidity and mortality in neonates worldwide, particularly in low-income countries. Identification of causative bacterial pathogens and assessment of their antimicrobial susceptibility are essential for guiding appropriate therapy and improving outcomes. The aim of this study was to determine the incidence, bacteriological profile and antibiotic susceptibility patterns of culture-positive EOS among a cohort of neonates in the Occupied Palestinian Territories (oPt). METHODS: This retrospective observational study was conducted on neonates with proven positive blood cultures or positive cerebrospinal fluid (CSF) admitted to eight neonatal intensive care units (NICU) in the West Bank, oPt between January 2017 and December 2019. Data on microbiology laboratory blood cultures were retrieved from NICU registers and medical records were reviewed to obtain data on mothers and neonates. RESULTS: Among the 95,319 neonates admitted to the eight NICUs during the study period, we detected 292 neonates with culture-proven EOS, resulting in an incidence rate of 3 per 1000 live births. The most common gram-positive bacteria identified among neonates were α hemolytic streptococcus (11.6%), CoNS (11.3%), and GBS (8.6%). E. coli (15.1%) and Klebsiella spp. (15.1%) were the most common gram-negative bacteria, followed by Acinetobacter (7.9%). Findings revealed gram-positive organisms were resistant to ciprofloxacin (57.1%) and highly sensitive to vancomycin (97.9%), meropenem (89.2%), amikacin (82.6%) and Piperacillin-Tazobactam (82.4%). Gram-negative organisms showed the highest antibiotic resistance to ampicillin (87.2%), cetofaxime, and highest sensitivity to meropenem (82.0%), Piperacillin-Tazobactam (70.7%), and amikacin (66.4%). CONCLUSION: Our findings underscore the importance of continuous surveillance of bacterial pathogens and their antimicrobial susceptibility patterns in the management of EOS among neonates in Palestinian hospitals. The findings generated will guide clinicians in selecting appropriate empirical therapies and facilitating early and targeted interventions. Future research should focus on strategies to enhance infection prevention and control measures in Palestinian neonatal care unites to mitigate the burden of EOS and antimicrobial resistance.