Background Infection prevention and control can be seen as a wicked public health problem as there is no consensus regarding problem definition and solution, multiple stakeholders with different needs and values are involved, and there is no clear end-point of the problem-solving process. Co-creation with stakeholders has been proposed as a suitable strategy to tackle wicked problems, yet little information and no clear step-by-step guide exist on how to do this. The objectives of this study were to develop a guideline to assist developers in tackling wicked problems using co-creation with stakeholders, and to apply this guideline to practice with an example case in the field of infection prevention and control. Methods A mixed-method approach consisting of the integration of both quantitative and qualitative research was used. Relevant stakeholders from the veterinary, human health, and public health sectors were identified using a literature scan, expert recommendations, and snowball sampling. The stakeholder salience approach was used to select key stakeholders based on 3 attributes: power, legitimacy, and urgency. Key values of stakeholders (N = 20) were derived by qualitative semi-structured interviews and quantitatively weighted and prioritized using an online survey. Results Our method showed that stakeholder identification and analysis are prerequisites for understanding the complex stakeholder network that characterizes wicked problems. A total of 73 stakeholders were identified of which 36 were selected as potential key stakeholders, and only one was seen as a definite stakeholder. In addition, deriving key stakeholder values is a necessity to gain insights into different problem definitions, solutions and needs stakeholders have regarding the wicked problem. Based on the methods used, we developed a step-by-step guideline for co-creation with stakeholders when tackling wicked problems. Conclusions The mixed-methods guideline presented here provides a systematic, transparent method to identify, analyze, and co-create with stakeholders, and to recognize and prioritize their values, problem definitions, and solutions in the context of wicked problems. This guideline consists of a general framework and although it was applied in an eHealth context, may be relevant outside of eHealth as well.