Objective: This study sought to investigate the faculty’s awareness, attitudes and use of open access, and the role of information professionals in supporting open access (OA) scholarly communication in Tanzanian health sciences universities. Methods: A cross-sectional survey was conducted. Semi-structured interviews were conducted with 16 librarians, while questionnaires were physically distributed to 415 faculty members in all eight Tanzanian health sciences universities, with a response rate of 71.1%. Results: The study found that most faculty members were aware about OA issues. However, the high level of OA awareness among faculty members did not translate into actual dissemination of faculty’s research outputs through OA web avenues. A small proportion of faculty’s research materials was made available as OA. Faculty were more engaged with OA journal publishing than with self-archiving practices. Senior faculty with proficient technical skills were more likely to use open access than junior faculty. Major barriers to OA usage were related to ICT infrastructure, awareness, skills, author-pay model, and copyright and plagiarism concerns. Interviews with librarians revealed that there was a strong support for promoting OA issues on campus; however, this positive support with various open access-related tasks did not translate into actual action. It is thus important for librarians and OA administrators to consider all these factors for effective implementation of OA projects in research and academic institutions. Conclusion: This is the first comprehensive and detailed study focusing on the health sciences faculty’s and librarians’ behaviours and perceptions of open access initiatives in Tanzania and reveals findings that are useful for planning and implementing open access initiatives in other institutions with similar conditions.