Nowadays, Nosocomial infection and hospital-acquired infection are a worldwide problem that increases morbidity and mortality among the hospitalized patients and surges an additional cost. Rural nurses have a paramount role in preventing infection and providing high quality of patient care. Aim: This study existed to assess the effectiveness of an in-service education program for improving nurses' performance concerning the application of infection control measures in rural hospitals. Setting: This study was conducted in a selected government rural hospital in Fayoum Governorate, Egypt. Sample: A purposive sample of 100 nurses working in the rural hospital were included. Tools: A structured interview guide was used to assess nurses' level of knowledge, and an observation checklist to assess their performance level before and after program implementation. Results: Nearly half of nurses had correct performance concerning infection control pre-program and improved to around 88.0% post-program implementation. There were statistically significant differences between nurse's total knowledge scales and subscales in pre and post-program. Likewise, there were highly statistically significant differences associated with nurses' correct performance score level pre and post-program. Conclusion: In-service education intervention reported incredible improvement in nurses' knowledge and performance toward infection control measures in the rural hospital. Recommendations: There is a desire for endless training courses for nurses to validate nurses' knowledge, performance regarding utilization of standard safeguards of infection control in the rural hospital. Finally, at the community level, the government has to consider the distribution of continuous education programs to expand all health services and resources in rural areas.