Background: Nurses who provide health care to people with Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) must be equipped with sufficient knowledge of Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome. The study aimed to evaluate the effect of educational intervention on nurses' knowledge, practice and attitudes regarding Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome. Methods: quasi-experimental design used and performed on convenient sample included 122 nurses working with Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome patients at Zagazig outpatient clinics of Fever and Chest Hospital. Three tools were used. Tool (I): self-administered structured questionnaire consisting of two parts about demographic characteristics of nurses and Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome knowledge, Tool (II): Observational Nurse’s Practice Checklist and Toll III regarding. Results, total satisfactory knowledge increased from 15.60% in pre-sessions to 84.40% in post sessions after one month and was reduced to 69.70% after three months. Also, nurses' satisfactory practice increased from 10.33±4.1 in pre-sessions to 14.45±3.1 in post sessions and was reduced to 13.26±2.9 after three months. Additionally, their positive attitude increased from 26.20% in pre-sessions to 72.10% in post sessions and reduced to 68.90% after three months. Conclusion: increasing nurses' knowledge, positive attitudes and good practices towards patients living with Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome. Recommendations: develop educational interventions for nurses’ focus on preventive methods, symptoms and modes of Human Immunodeficiency Virus transmission.