Objective: To verify the effect of the educational intervention TIV-Disinfect (Intravenous Therapy-Disinfect) on the knowledge of nursing professionals in pediatric inpatient units. Method: Quasi-experimental study, with a dependent sample of 65 nursing professionals from pediatric inpatient units. A questionnaire with objective questions regarding the disinfection of venous devices was used, and pre- and post-tests were applied. Data collection was conducted in two public hospitals in southern Brazil, between April and July 2022. For data analysis, descriptive statistics and McNemar's test were used. Results: There was a statistically significant difference between the pre- and post-tests regarding variables of passive disinfection, and recommended antiseptics and materials, rubbing and drying. A lower frequency of incorrect alternatives was noted after the intervention. Conclusion: The educational intervention had a positive effect. Better results were observed in the knowledge of nursing professionals regarding the disinfection of venous devices, as well as allowing the professionals to reinforce already consolidated knowledge. This intervention can be used as a tool for updating and/or continuing training of nursing professionals, aiming at the safety of pediatric patients and care practices.