Background: Nurses and midwives are central to the implementation and delivery of quality care through evidence-based practice (EBP). However, implementation of EBP in nursing and midwifery is under-researched with few examples of systematic and sustained change. The Registered Nurses Association of Ontario’s Best-Practice Spotlight Organization (BPSO) Program was adopted in South Australia as a framework to systematically implement EBP in two diverse and complex healthcare settings. Methods: The study was a post-implementation, mixed-method evaluation conducted at two healthcare settings in Adelaide, South Australia utilizing qualitative and quantitative data. Proctor’s implementation evaluation framework guided the evaluation design. Information sources included; interviews, focus groups, questionnaires, and document review. Results: Clinical and executive staff (n = 109 participants) from a broad range of stakeholder groups participated in the interviews, focus groups, and returned questionnaires. A number of facilitators directly affecting program implementation were identified; these pertained to embedding continuity into the program’s implementation and delivery, a robust governance structure, and executive sponsorship. Barriers to implementation were also identified. These barriers pertained to organizational or workforce challenges; staff turnover and movement (e.g., secondment), insufficient staff to allow people to attend training, and a lack of organizational commitment to the program, especially at an executive level. As a result of successful implementation, it was observed that over three years, the BPSO program positively influenced the uptake and implementation of EBP by clinicians and the organizations into which they were introduced. Conclusions: The BPSO model can be translocated to new healthcare systems and has the potential to act as a mechanism for establishing and sustaining EBP change. This study was the first to apply an implementation evaluation framework to the BPSO program, which allowed for structured analysis of facilitating or impeding factors that affected implementation success. The findings have important implications for other health systems looking to translocate the same or similar EBP programs, as well as contributing to the growing body of implementation evaluation literature.