Climate change, biodiversity loss and large-scale environmental degradation are widely recognized as the biggest health threats of the 21st century, with the African continent already amongst the most severely affected and vulnerable to their further progression. The healthcare system’s contribution to climate change and environmental degradation requires healthcare professionals to address environmental issues urgently. However, the foundation for context-relevant interventions across research, practice, and education is not readily available. Therefore, we conducted a convergent mixed-methods study to investigate South African healthcare professionals’ knowledge, attitudes, practices, and barriers to environmental sustainability. Healthcare professionals participated in a cross-sectional questionnaire (n = 100) and in-depth semi-structured focus group discussions (n = 18). Data were analyzed using descriptive statistics and thematic analysis, respectively, and integrated to provide holistic findings. Our results confirm overwhelmingly positive attitudes and a high degree of interest in education, implementation, and taking on more corresponding responsibility, but a lack of substantial knowledge of the subject matter, and only tentative implementation of practices. Identified barriers include a lack of knowledge, resources, and policies. Further research, education, and policy development on overcoming these barriers is required. This will facilitate harnessing the extant enthusiasm and advance environmental sustainability in South Africa’s healthcare practice.