The mediation of guided visits is a crucial factor in achieving the educational potential of non-formal science education settings. Mediators must mobilise various types of knowledge to shape their educational practices and enhance visitors’ experiences. Thus, the aim of this study was to characterise the stages of professional development of science centre, museum, aquarium, botanical garden, and zoo mediators. The study identified the perception of mediators regarding the development of their professional knowledge over time based on the application of a questionnaire to 78 mediators from different non-formal settings in Brazil and in-depth interviews with 8 of them. The study identified three stages of professional development for mediators: insertion, stabilization and diversification, which characterize the knowledge that mediators perceive as more or less developed in each stage. The potential impact of these stages on the educational practices of mediators and, subsequently, on visitors’ experience was discussed.