Information on the impact of care bundles has been mainly acquired in adult intensive care units (ICUs). However, specific data for educational programs are scarce. Our objective was to analyze the impact of an educational program on the knowledge and prevention of catheter-related bloodstream infection (CRBSI) in two pediatric intensive care units (P-ICUs). A prospective study was carried out at a large teaching institution in Madrid, Spain. Healthcare workers' (HCWs) knowledge of guidelines for the prevention of CRBSI was assessed before and after the educational program using a questionnaire covering 12 issues. A 20-min program was offered to all HCWs on each ICU shift. The incidence density of CRBSI was assessed before, during, and after the educational program. A total of 174 questionnaires were completed by HCWs from both the neonatal ICU (N-ICU) and the P-ICU before the intervention and 54 were completed after the intervention (120 participants were not present during this period). The incidence density of CRBSI before, during, and after the intervention was 6.2, 5.2, and 9.3 in the N-ICU and 2.2, 3.1, and 2.9 in the P-ICU (p > 0.05). A single 20-min educational intervention on the prevention of CRBSI significantly improved HCWs' knowledge, but was not enough to reduce the incidence density of CRBSI.