Objectives: e-Bug, a junior and senior school educational programme to decrease the spread of infection and unnecessary antibiotic use, was developed and consisted of eight sections providing information on the spread, treatment and prevention of infection as well as basic information on microbes, both useful and harmful. Each section comprised teacher background information, lesson plans and an interactive student activity, and extension activities were also available for more able students. This study aimed to evaluate the effectiveness of the e-Bug pack in improving children's knowledge in these key areas, when used within the National Curriculum in England, France and the Czech Republic. Methods: Junior (9-11 years) and senior (12-15 years) school classes were divided into either control or intervention groups for evaluation of the resource. Students were required to complete identical knowledge questionnaires at three timepoints (before, immediately after and 6 weeks after teaching), to assess knowledge change and retention. Teaching, using the e-Bug pack, was given by junior and senior school teachers. Results: The junior e-Bug teaching pack demonstrated a significant improvement in student's knowledge in all sections and there was no significant decrease in student knowledge observed after a 6 week period. Knowledge improvement with the senior e-Bug pack varied between regions, although consistent improvement was observed for Gloucestershire (England) and Ostrava (Czech Republic). Conclusions: Although a success, modifications are required in both packs to further improve student knowledge and make the packs more appealing.