Background: Acquired immunodeficiency syndrome (AIDS) is a common challenge that hospitals face globally. This study aimed to examine the impact of Educational Program on Acquired Immunodeficiency Syndrome Prevention among Nurses. Materials and Methods: This quasi-experimental study was conducted at three hospitals (Wad Madani Teaching Hospital, Obstetrics and Gynecology Teaching Hospital, and Gezira Hospital for Renal Diseases and Surgery) in Wad Madani City, Gezira State, Sudan. Targeted all nurses present during the study period at the three hospitals. Data were obtained through a modified questionnaire and observational checklist created by the authors. Results: The mean (±standard deviation) total knowledge score was 14.35 (±6.51) before implementation of the educational program, as compared to 30.61 (±6.32) after the program (T-value:–5.6, p-value: 0.006, level of significance:<0.05). The mean total attitude score was 6.4 (±6.20) before implementation of the training program, as compared to 11.7 (±5.56) after the program (T-value:–6.5, p-value: 0.004). The mean total practice score was 9.31 (±6.45) before implementation of the educational course, as compared to 21.70 (±6.86) after the program (T-value:–13.35, p-value: 0.001). Conclusions and Recommendations: Results showed that the intervention program improved the HIV/AIDS knowledge and practices of the participants. Further AIDS education and training programs should be mandatory for all nurses.