Background: The internship year is a valuable prospect for new nurses to advance their practice in the real world setting. However, nurse interns are meeting a variety of stressors that could hinder their work outcomes and even their overall wellbeing. Therefore, assertiveness training is required to improve their self-esteem and decrease stress associated with the demanding nature of the nursing profession. The present study aimed to assess the effect of assertiveness training program on nurse interns' self-esteem and stress. A quasi-experimental design was followed to achieve the aim of the present study. Setting: It was carried out at El-Fayoum University Hospitals. Subjects and methods: The study subjects included all the available nurse interns who were enrolled at the academic year 2016-2017. Three tools were used to collect data for this study, the assertive behavior in nurses' questionnaire, the self-esteem scale, and the perceived stress scale. Results revealed that there was a significant improvement in nurse interns' assertiveness mean score after the program implementation (P < 0.05). As well, there was statistically insignificant enhancement in their self-esteem mean score, where P- value > 0.05. Additionally, there was a statistically significant reduction in their total mean score of stress after the program implementation (P < 0.01). Conclusion: The assertiveness training is beneficial in developing nurse interns' self-esteem and decreasing stress. Therefore, it is recommended to provide assertiveness training to the non-assertive nurse interns at the beginning of the internship year to build nurse interns' self-concept and self-esteem and subsequently decrease stress.