In this article we present the development of a new, web-based, graphical authentication mechanism called ImagePass. The authentication mechanism introduces a novel feature based on one-time passwords that increases the security of the system without compromising its usability. Regarding usability, we explore the users’ perception of recognition-based, graphical authentication mechanisms in a web environment. Specifically, we investigate whether the memorability of recognition-based authentication keys is influenced by image content. We also examine how the frequency of use affects the usability of the system and whether user training via mnemonic instructions improves the graphical password recognition rate. The design and development process of the proposed system began with a study that assessed how the users remember abstract, face or single-object images, and showed that single-object images have a higher memorability rate. We then proceeded with the design and development of a recognition-based graphical authentication mechanism, ImagePass, which uses single-objects as the image content and follows usable security guidelines. To conclude the research, in a follow-up study we evaluated the performance of 151 participants under different conditions. We discovered that the frequency of use had a great impact on users’ performance, while the users’ gender had a limited task-specific effect. In contrast, user training through mnemonic instructions showed no differences in the users’ authentication metrics. However, a post-study, focus-group analysis revealed that these instructions greatly influenced the users’ perception for memorability and the usability of the graphical authentication. In general, the results of these studies suggest that single-object graphical authentication can be a complementary replacement for traditional passwords, especially in ubiquitous environments and mobile devices.