Klebsiella pneumoniae is a frequent cause of infectious outbreaks in Neonatal Intensive Care Units (NICUs). The aim of this paper is to describe an outbreak occurred in a 13-bed NICU and the control measures adopted in order to interrupt the chain of transmission. We described the microbiological investigations, the NICU staff compliance to the infection control measures by means of a specifically designed check-list and the control measures adopted. Six cases of primary bloodstream infections sustained by ampicillin/piperacillin-resistant Klebsiella pneumoniae were observed over a two-month period. One culture obtained from a 12% saccarose multiple-dose solution allowed the growth of Klebsiella pneumoniae. During the inspections performed by the Hospital Infection Control Team, using the check-list for the evaluation of the NICU staff compliance to the infection control measures, several breaches in the infection control policy were identified and control measures were adopted. In our case the definition of a specific check-list led to the adoption of the correct control measures. Further studies would be helpful in order to develop a standard check-list able to identify critical flows in the adhesion to the guidelines. It could be used in different NICUs and allow to obtain reproducible levels of infection control.