This study aimed to determine the distribution of bacterial and fungal pathogens in neonatal sepsis cases in a neonatal unit during the COVID-19 pandemic and to investigate antimicrobial susceptibility/resistance profiles using phenotypic and molecular methods. A total of 940 samples were analysed in this study. Pathogens were identified using conventional culture methods and the Siemens MicroScan Walkaway 96 Plus system. Antimicrobial susceptibility of the isolates was determined phenotypically using the Siemens MicroScan Walkaway 96 Plus system and by PCR. Selected resistance genes were detected using multiplex PCR. A total of 113 isolates were recovered, predominantly Gram-negative bacilli (72%). The most frequent pathogens were coagulase-negative staphylococci (n = 28), Acinetobacter baumannii/haemolyticus (n = 22), Klebsiella spp. (n = 16), Escherichia coli (n = 10), Enterococcus spp. (n = 10), Candida albicans (n = 8), Stenotrophomonas maltophilia (n = 6), Serratia marcescens (n = 5), Staphylococcus aureus (n = 5) and Pseudomonas aeruginosa (n = 3). High resistance rates to beta-lactam antibiotics were observed, particularly among Gram-negative isolates. ESBL production ranged from 33%-50% in Enterobacterales, while MDR rates varied between 20% and 95.45% across species. All C. albicans isolates were susceptible to amphotericin B and to caspofungin. These findings highlight the urgent need for strengthened infection control measures and tailored empirical therapy.