Background: Ambidexterity is an emerging concept in nursing management that holds promise for achieving remarkable outcomes. To encourage ambidextrous behaviors among nurse managers, it is crucial to implement an ambidexterity training program. Aim: This study aimed to investigate the effect of an ambidexterity training program on the innovative behavior and leadership competencies among nurse managers. Subjects and Methods: This study employed a pre-experimental, single-group pre- and post-test research design that involved all 47 nurse managers from three hospitals in Port Said, Egypt: Port Said General Hospital, El-Zohuor Hospital, and Port Fouad General Hospital. Participants received a program of a four 120-min training sessions delivered over a 2-day workshop. The Ambidexterity Knowledge Questionnaire, the Ambidexterity Behavior Scale, Innovative Behavior Inventory, and the Penn State Leadership Competency Inventory were administered at three time points: pre-training, two weeks post-training, and 3-month follow-up. Single-factor repeated measures ANOVA was used to compare outcomes across the three time points. Results: Compared to pre-training scores, participants who underwent the ambidexterity training demonstrated significant improvement in their ambidexterity knowledge (F = 116.14, p < 0.001), ambidexterity behaviors (F = 12.74, p < 0.001), innovative behavior (F = 29.69, p < 0.001), and leadership competencies (F = 21.88, p < 0.001) at both post-training and 3-month follow-up assessments. While participants maintained the improvement at the 3-month follow-up assessment compared to pre-training, the degree of improvement was lower than that observed post-training. Conclusion: The ambidexterity training program was effective in increasing nurse managers' ambidexterity behavior, innovative behavior, and leadership competencies. Recommendations: Hospital administrators should consider incorporating ambidexterity training as part of the professional development programs offered to nurse managers.