BACKGROUND: Antimicrobial resistance (AMR) in neonatal intensive care units (NICUs) complicates treatment of healthcare-associated infections, causing high morbidity and mortality, especially among low-birth-weight and critically ill infants. This study evaluates AMR prevalence, risk factors, outcomes and infection control measures at Felege Hiwot Hospital, aiming to guide clinical practices, antimicrobial stewardship, and improved neonatal health outcomes. METHODS: This mixed-methods study (Oct 2022-Jun 2023) assessed AMR in NICU neonates by analyzing 420 blood samples, environmental swabs, and staff insights. Bivariable and multivariable regressions identified significant variables, and content analysis was used for qualitative data. RESULTS: Out of 420 samples, 35% tested positive for AMR pathogens, with Coagulase-Negative Staphylococci (16.7%), Klebsiella pneumoniae (12.9%), and Acinetobacter spp. (5.6%) being the most prevalent. Resistance rates for Gentamicin, Cotrimoxazole, and Ciprofloxacin were alarmingly high (98-100%), while Amikacin demonstrated low resistance (3.0-5.56%), indicating potential efficacy. Among the neonates admitted, 91.8% survived, and 8.2% succumbed. Risk factor analysis revealed that improper PPE usage (AOR 3.90, p <
0.001), non-functional handwashing sinks (AOR 3.20, p <
0.001), and inadequate disinfection practices (AOR 2.70, p <
0.001) were strongly associated with microbial contamination. Environmental factors, including cockroach presence (AOR 1.80, p = 0.040) and high traffic flow (AOR 2.10, p = 0.005), were also significant contributors. The qualitative data analysis confirmed that improper PPE use, inadequate disinfection practices, pest control challenges, and non-functional handwashing sinks significantly contributed to microbial contamination risks in the NICU, aligning with the quantitative findings. CONCLUSIONS: This study underscores key factors driving AMR in NICUs, such as inadequate IPC practices and environmental contamination, alongside high resistance to Cotrimoxazole and Ciprofloxacin. Amikacin shows promise as an effective treatment for CONS. Urgent actions, including strengthened IPC measures, staff training, and environmental management, are crucial to combat AMR, ensuring improved neonatal care and outcomes.