OBJECTIVES: To evaluate the effectiveness of a web-based instruction in the interpretation of anatomy in images acquired with maxillofacial cone beam CT (CBCT). METHODS: An interactive web-based education course for the interpretation of craniofacial CBCT images was recently developed at our institution. Self-evaluation modules on correlative anatomical features were also included to support the learning process. Three e-learner groups were selected to evaluate the effectiveness of the educational modules. The three groups were (1) oral health specialists (OHSs) (comprising periodontologists, prosthodontists, orthodontists and maxillofacial surgeons); (2) third grade (DS3) and (3) first grade (DS1) undergraduate dental students. The assessment modules that were part of the interactive web-course content were administered after delivery of the course material. In addition, each group received a computer affinity questionnaire to quantify the extent of knowledge about computers and a perception questionnaire to assess their attitudes toward the web-course. RESULTS: The OHS group yielded significantly better scoring results in the post-course test than the pre-course test. However, no statistically significant differences in test scores were found for both undergraduate student groups (DS1 and DS3). All groups presented a highly positive attitude towards the web-course, as was demonstrated by the post-course perception questionnaire. CONCLUSIONS: The present CBCT educational course is an effective didactic method for teaching OHSs the anatomical interpretation of CBCT multiplanar reformatted images and, for undergraduate students, it was found to be as effective as conventional educational methods in dentistry. The efficacy of a web-based educational course requires further evaluation.