Background: Antibiotic resistance is an emerging worldwide concern with serious repercussions in terms of morbi-mortality. Aim of study: Was to evaluate the effect of educational guidelines regarding preventive measures of antibiotic resistance on nursing students' knowledge and attitude. Research design: Quasi-experimental research design (pre-test and post-test) was utilized to achieve the study's aim. Setting: The study was conducted at Faculty of Nursing, Benha University, Benha, Egypt. Sample: Simple random sample of 1 st year nursing students at Benha Faculty of Nursing were taken, from both sexes, during the second term of the academic year 2020/2021, the total sample included 263 students. Tools of data collection: Two tools were used; I: Structured interviewing questionnaire that included socio demographic and knowledge parts and II: Students' attitude scale regarding antibiotic use. Results: There was a highly significant statistical difference in total knowledge level throughout different study periods (pre, immediately post and after one month) of implementing educational guidelines. Also, there was a highly significant statistical difference in total attitude level regarding antibiotic use throughout different study periods. Finally, there was a highly significant statistical positive correlation between students' total knowledge and total attitude after implementing educational guidelines; during immediately post and after one month periods where the higher the level of knowledge, the more positive is the attitude. Conclusion: Nursing students' knowledge and attitude regarding antibiotic use and resistance were improved significantly after implementation of educational guidelines. Recommendations: Ongoing educational programs regarding antibiotic use & resistance and integrating brief content about that topic in first year educational curricula are needed to improve nursing students' knowledge and attitude.