Osmosis and diffusion are fundamental topics in science education because many processes in nature largely depend on these phenomena. However, many students usually hold misconceptions and have a lack of knowledge about these topics. This study explores pre-service teachers’ understanding of osmosis and diffusion in relation to their prior formal education. The main reason for the study was the need to support pre-service teachers so that they can effectively teach these topics. A total of 243 first-year students in bachelor’s programs at the University of Ljubljana participated in the study. The results showed that students that graduated from vocational and technical secondary schools had a weaker understanding of these topics compared to students from general secondary schools. Furthermore, only students that had passed the leaving exam in biology showed a better understanding of osmosis and diffusion.