Background: Intravenous therapy is an important part of the treatment of many hospitalized neonates. Intravenous solutions are medications. Administration of medications is part of the controlled acts authorized to nursing. This is the nurse's responsibility to ensure that the neonate receives the ordered solution and additives at the ordered rate. Aim: This study aimed to assess the neonatal nurses' knowledge and practice regarding applying safety measures during neonates' intravenous infusion. Research design: A descriptive study design applied in the current study. Setting: The study conducted at the Neonatal Intensive Care Unit at Minya Al-Qamh General Hospital affiliated to the Ministry of Health and Population and Al-Saadian Central Hospital affiliated to the Ministry of Health and Population. Study subjects: A convenient sample was included all nurses available in the previously mentioned setting regardless of their age, gender, qualifications, years of experience or any training courses regarding intravenous therapy. A purposive sample of neonates with inclusions criteria: Preterm neonates, low birth weight, normal birth weight and neonates with fluid and electrolyte imbalance. Total subjects number of 70 nurses and 70 neonates.  Tools: Data collection obtained by using the following tools: Tool 1: Pre-designed interviewing questionnaire to assess nurse's knowledge, Tool (2): Observational checklist to assess nurse's practice. Results: The study found that, near a half of the studied nurses had a good level of knowledge regarding applying safety measures during neonates' intravenous infusion, while more than one quarter had average level and less than one quarter had poor level of knowledge. Also, the majority of the studied nurses had competent total practice level. Conclusion: The study concluded that there are statistically significant positive correlations between the nurses' total scores of knowledge and practice regarding applying safety measures during neonates' intravenous infusion. Recommendations: Establish continuous training courses to improve neonatal nurses' knowledge and practices about applying safety measures during neonates' intravenous infusion.