Purpose : This study aimed to compare the knowledge, attitude, and practice related to handwashing among undergraduate students majoring and not majoring in health science. Methods : This descriptive study included 188 undergraduate students with 100 majoring and 88 not majoring in health science. Data were collected using a structured questionnaire and they were analyzed with the IBM SPSS 24.0 program using descriptive statistics, t-tests, Pearson’s correlation coefficients and a multiple regression. Results : Mean scores on knowledge and attitude toward handwashing were 13.86(±1.56) and 21.05(±2.34) respectively in students majoring health science, whereas the same were 13.22(±1.73) and 19.76(±2.24) in students not majoring health science respectively. In both groups, there was significant correlations between knowledge and attitude(r=.32, p<.001; r=.33, p=.002), and between attitude and practice (r=.27, p=.006; r=.40, p<.001) respectively. Attitude toward handwashing (=0.20, p=.028), experience of education about handwashing (=0.33, p<.001) and perception about health status (=-0.23, p=.014) were significant predictors of handwashing practice in students majoring health science. On the other hand, only attitude toward handwashing (=0.37, p<.001) was a significant predictor of handwashing practice in students not majoring health science. Conclusion : The present results reveal the importance continuing various educational programs on handwashing and the need to identify their longitudinal effects to improve handwashing practice among students majoring in health science. Additionally, when developing strategies to improve handwashing habits, we need to consider factors that could help change attitudes toward handwashing.