Purpose: This study was done to develop a web-based education program on appropriate antibiotic use and test the effects of the program on knowledge and attitudes towards antibiotic use in Korean adolescents. Methods: The web-based education program was developed through an extensive literature review and professional advisory meetings including technical assistance from a web-based education programmer and content experts. A convenience sample of 851 students from middle and high schools participated in the assessment of effects of the program. Knowledge and attitudes of the students towards antibiotic use and satisfaction with the web-based education program were measured. Descriptive statistics and paired t-test were used to analyze the data. Results: There were significant improvements in knowledge and attitudes towards antibiotic use following self-learning via the web-based education program in both middle and high school students. High school students demonstrated higher scores in knowledge and attitudes than middle school students. Conclusion: The results of this study indicate that this web-based education program on appropriate antibiotic use is a convenient and effective medium for self-learning in adolescents. Therefore the web-based program should be put into wide use as an effective and convenient teaching method for health education in secondary schools.