The provision of probiotics benefits the health of a wide range of organisms, from humans to animals and plants. Probiotics can enhance stress resilience of endangered organisms, many of which are critically threatened by anthropogenic impacts. The use of so-called ‘probiotics for wildlife’ is a nascent application, and the field needs to reflect on standards for its development, testing, validation, risk assessment, and deployment. Here, we identify the main challenges of this emerging intervention and provide a roadmap to validate the effectiveness of wildlife probiotics. We cover the essential use of inert negative controls in trials and the investigation of the probiotic mechanisms of action. We also suggest alternative microbial therapies that could be tested in parallel with the probiotic application. Our recommendations align approaches used for humans, aquaculture, and plants to the emerging concept and use of probiotics for wildlife.