Background Motivational interviewing (MI) is an established communication method for enhancing intrinsic motivation for changing health behavior. E-learning can reduce the cost and time involved in providing continuing education and can be easily integrated into individual working arrangements and the daily routines of medical professionals. Thus, a Web-based course was devised to familiarize health professionals with different levels of education and expertise with MI techniques for patients with chronic conditions. Objective The aim of this study was to report participants’ opinion on the practicality of MI (as learned in the course) in daily practice, stratified by the level of education. Methods Participants (N=607) of the MI Web-based training course evaluated the course over 18 months, using a self-administered questionnaire. The evaluation was analyzed descriptively and stratified for the level of education (medical students, physicians in specialist training [PSTs], and general practitioners [GPs]). Results Participants rated the applicability of the skills and knowledge gained by the course as positive (medical students: 94% [79/84] good; PSTs: 88.6% [109/123] excellent; and GPs: 51.3% [182/355] excellent). When asked whether they envisage the use of MI in the future, 79% (67/84) of the students stated to a certain extent, 88.6% (109/123) of the PSTs stated to a great extent, and 38.6% (137/355) of GPs stated to a great extent. Participants acknowledged an improvement of communication skills such as inviting (medical students: 85% [72/84]; PSTs: 90.2% [111/123]; GPs: 37.2% [132/355]) and encouraging (medical students: 81% [68/84]; PSTs: 45.5% [56/123]; GPs: 36.3% [129/355]) patients to talk about behavior change and conveying respect for patient’s choices (medical students: 72% [61/84]; PSTs: 50.0% [61/123]; GPs: 23.4% [83/355]). Conclusions Participants confirmed the practicality of MI. However, the extent to which the practicality of MI was acknowledged as well as its expected benefits depended on the individual’s level of education/expertise.