Despite the fact that a large number of web applications are used in the medical community, there are still certain technological challenges that need to be addressed, for example, browser plug-ins and efficient 3D visualization. These problems make it necessary for a specific browser plug-in to be preinstalled on the client side when launching applications. Otherwise, the applications fail to run due to the lack of the required software. This paper presents the latest techniques in hypertext markup language 5 (HTML5) and web graphics library (WebGL) for solving these problems and an evaluation of the suitability of the combination of HTML5 and WebGL for the development of web-based medical imaging applications. In this study, a comprehensive medical imaging application was developed using HTML5 and WebGL. This application connects to the medical image server, runs on a standard personal computer (PC), and is easily accessible via a standard web browser. The several functions required for radiological interpretation were implemented, for example, navigation, magnification, windowing, and fly-through. The HTML5-based medical imaging application was tested on major browsers and different operating systems over a local area network (LAN) and a wide area network (WAN). The experimental results revealed that this application successfully performed two-dimensional (2D) and three-dimensional (3D) functions on different PCs over the LAN and WAN. Moreover, it demonstrated an excellent performance for remote access users, especially over a short time period for 3D visualization and a real-time fly-through navigation. The results of the study demonstrate that HTML5 and WebGL combination is suitable for the development of medical imaging applications. Moreover, the advantages and limitations of these technologies are discussed in this paper.