This study explored opt-out HIV testing in an Australian general practice. The aims were to: (1) determine the effect of the opt-out approach on the number of HIV tests performed; and (2) explore the acceptability of opt-out HIV testing from the healthcare providers' perspective. A prospective mixed-methods study of opt-out HIV testing over a 2-year period (March 2014-March 2016) was conducted. Implementation was based on a theoretical framework that was developed specifically for this study. The setting was Homeless Healthcare, a health service in Perth, Western Australia. The number of HIV tests conducted during the control year (usual practice) was compared with the intervention year (opt-out testing). After the intervention, the healthcare providers (n≤8) were interviewed about their experiences with opt-out HIV testing. Directed content analysis was used to explore the qualitative data. HIV testing rates were low during both the control year and the intervention year (315 HIV tests (12% of the patient cohort) and 344 HIV tests (10%) respectively). Opt-out HIV testing was feasible and acceptable to the participating healthcare providers. Other health services could consider opt-out HIV testing for their patients to identify people with undiagnosed infections and sustain Australia's low HIV prevalence.