The current study aimed to assess the effect of interprofessional education on participants' self-efficacy perception of interprofessional communication at different levels of a healthcare system (relationships in the interpersonal, interprofessional, and administration levels). This is a sequential explanatory mixed-method study. Learners include surgical residents, surgical technology nurses, and anesthesia nurses (n = 130) who participated in the intervention and control groups. The quasi-experimental study was conducted in the first step. The educational strategy was interprofessional education and the main teaching method was case-based learning. The self-efficacy perception of the participants was assessed by a 26-item questionnaire before and 1 month after the intervention. The descriptive tests (mean, SD, percentage), student t-tests, and ANOVA were analyzed. The participants' experiences were explored through semi-structured interviews. Qualitative content was analyzed by Lundman and Graneheim's approach. The results showed that the scores of the learners in the intervention group (4.05 ± 0.31) improved significantly compared to the control group (3.35 ± 0.37) (p = 0.0001). In the present study, the experiences of the participants were categorized into a theme "explicit and implicit confrontations with perceived distrust in the system" which includes two categories "failure to internalize professionalism among team members" and "dominance of a paternalistic culture within the health care system". The results showed the positive impact of interprofessional education interventions on participants' self-efficacy perception of interprofessional communication. The results showed that interprofessional education, with the participation of different professionals and managers at different levels of a healthcare system, improved the participants' perception of self-efficacy regarding interprofessional communication. The results showed the improvement in the participants' communication with team members and managers required to solve the perceived explicit and implicit confrontations in the surgical departments. The implementation of an interprofessional education strategy focused on interprofessional professionalism and collaboration principles in clinical activities, formal and informal education in the surgical department is recommended.