In-service education for oncology nurses usually adopts didactic teaching. This study investigated the effects of virtual reality (VR) and a digital learning-based Port-A-catheter educational course for oncology nurses. A mixed-methods research design was employed, with a convenience sample of 43 nurses from a regional teaching hospital in Taiwan participating. Measurements were taken at three time points: pre-test, 1st post-test, and 2nd post-test. The data was analyzed using descriptive statistics and repeated ANOVA tests. Results showed significant improvement in Port-A-catheter knowledge and skill levels (p < 0.0001) and high learning attitude and satisfaction scores of 4.29 ± 0.46 and 4.31 ± 0.58 points, respectively. Five qualitative themes emerged, highlighting the realistic VR scenarios, VR practice’s usefulness, willingness to learn with VR, VR system limitations, and the potential for future courses. The study concluded that a VR-based educational course effectively enhanced nurses’ knowledge, skills, learning attitude, and satisfaction, recommending the inclusion of diverse clinical scenarios for practical learning.