Central line-associated bloodstream infection (CLABSI) is a significant cause of morbidity and mortality in neonatal intensive care units (NICUs). A “bundle” is defined as a combination of evidence-based interventions that provided they are followed collectively and reliably, are proven to improve patient outcomes. The aim of this quasi-experimental study was to assess the impact of new central line insertion, dressing, and maintenance “bundles” on the rate of CLABSI and catheter-related complications. We performed a quality improvement (QI), prospective, before-after study. In the first 9-month period, the old “bundles” and pre-existing materials were used/applied. An intervention period then occurred with changes made to materials used and the implementation of new “bundles” related to various aspects of central lines care. A second 6-month period was then assessed and the CLABSI rates were measured in the NICU pre- and post-intervention period. The QI measures were the rate of CLABSI and catheter-related complications. Data are still being collected after the study to verify sustainability. The implementation of the new “bundles” and the change of certain materials resulted in a significantly decreased rate of CLABSI (8.4 to 1.8 infections per 1000 central venous catheter (CVC) days, p = 0.02,) as well as decreased catheter-related complications (47 to 10, p < 0.007).Conclusions: The analysis of pre-existing “bundles” and the implementation of updated central line “bundles” based on best practice recommendations are crucial for reducing the rate of CLABSI in the NICU. The implementation of the new evidence-based central line “bundles” was associated with a significant reduction in CLABSI rate in our unit soon after implementation. What is Known: • Central line-associated bloodstream infection (CLABSI) is a major cause of morbidity and mortality in the neonatal population. • The implementation of evidence-based “bundles” in the NICU is associated with a reduction in the incidence of CLABSI. What is New: • For the improvement in quality care in the NICU, audits are necessary to assess the existing systems. • The “Plan-Do-Study-Act cycle” is an effective tool to use when tackling challenges in an existing system. Using this tool assisted in the approach to reducing CLABSI in our NICU.