Purpose: This single-group pre-post experimental study aimed to develop and apply a simulation-based infection control education program for neonatal intensive care unit (NICU) nurses to verify its effectiveness and provide baseline educational materials to strengthen NICU nurses’ infection control competencies. Methods: The educational program was developed according to the five phases of analysis, design, development, implementation, and evaluation models, with scenarios based on the educational needs of NICU nurses. To verify the program’s effectiveness, a study was conducted on 28 NICU nurses of general and tertiary hospitals. NICU nurses’ infection control knowledge, performance confidence, critical thinking disposition, and satisfaction with the education were surveyed after applying the education program. Results: The simulation-based NICU infection control educational program was effective for NICU nurses’ infection control knowledge (t=−7.35, p < .001), performance confidence (t=−6.30, p < .001), and critical thinking disposition (t=−5.12, p < .001). Conclusions: The infection control educational program can contribute to infection prevention and patient safety by improving the NICU nurses' infection control competencies and be used as a baseline educational material for infection control education.