Central venous line (CVL) is a necessary device used frequently in neonatal intensive care units (NICUs) for critically ill neonates who need continuous monitoring and resuscitation treatment. Significant evidence has accumulated indicating that CVL nursing care bundle is considered as an essential component of CVL infection prevention practice to combat the incidence of CVL infection. Aim: This study aimed to examine the effect of applying nursing care bundle on controlling central venous line infection in neonatal intensive care units. Subject and method: A quasi-experimental design was used. The study was conducted at NICU at Kafr El Sheikh General Hospital. The study included a convenience sample of all available bedside/ working nurses (n= 30), 79 preterm infant (recruited for 3 months' period). Tools: Three tools were used to collect data. Tool 1: An interview questionnaire for nurse's knowledge. Tool 2: Observational checklist used to assess nurses' performance regarding CVL care bundle. Tool 3: Laboratory investigations tool for preterm infants. Results: More than half of studied nurses (56.7%) didn't receive any training program about CVL care bundle for controlling CVL infection in NICU and the majority of the studied nurses had insufficient knowledge, as well as the majority of the studied nurses, had an unsatisfactory practice regarding CVL care bundle preprogram. The sufficient knowledge level and satisfactory practices had improved immediately post and 3 months post program implementation. About half (51,9%) had negative CRP post-program implementation, while (54,4%) of neonates had negative blood culture post-program. Conclusion: The program had a positive effect on the NICU nurses' knowledge & practices immediately post& post 3 months of the program implementation. Recommendation: Including CVL nursing care bundle practices into nurses' routine care and developing regular and continuous educational programs for the nurses in NICU according to their needs aiming to upgrading their knowledge and improving their practice for critically ill neonates.