EUDONORGAN, a European Union-funded project to improve organ and tissue donation, included a blended-based “Train the Trainers” program, which was implemented with the support of an international consortium from Croatia, Italy, Slovenia, and Spain. The web-based training included seven modules for which medical aspects, educational tips, and practical activities were scored using a 5-point Likert scale. The overall mean scores of satisfaction were higher than 4 for each module, without significant differences between HCPs and OKPs. In the face-to-face training survey similar scores above 4 were obtained for most items. Knowledge acquisition improved significantly in both HCPs and OKPs, as well as in transplant/donor coordinators, medical doctors, registered nurses, anesthesiologists/intensivists, and intensive care nurses. Improvements in attitudes and perceptions regarding organ donation were also observed, particularly among HCPs. In the accomplishment of the learning process, a successful pass mark of 95% was obtained. The “Train the Trainers” program was associated with an improvement in learning and attitudes of healthcare and non-healthcare professionals for the benefit of organ and tissue donation.