Background: e-Bug is a pan-European educational resource for junior and senior school children, which contains activities covering prudent antibiotic use and the spread, treatment, and prevention of infection. Teaching resources for children aged 7-15 years are complemented by a student website that hosts games and interactive activities for the children to continue their learning at home. Objective: The aim of this study was to appraise young people’s opinions of 3 antibiotic games on the e-Bug student website, exploring children’s views and suggestions for improvements, and analyzing change in their knowledge around the learning outcomes covered. The 3 games selected for evaluation all contained elements and learning outcomes relating to antibiotics, the correct use of antibiotics, and bacteria and viruses. Methods: A mixed methodological approach was undertaken, wherein 153 pupils aged 9-11 years in primary schools and summer schools in the Bristol and Gloucestershire area completed a questionnaire with antibiotic and microbe questions, before and after playing 3 e-Bug games for a total of 15 minutes. The after questionnaire also contained open-ended and Likert scale questions. In addition, 6 focus groups with 48 students and think-aloud sessions with 4 students who had all played the games were performed. Results: The questionnaire data showed a significant increase in knowledge for 2 out of 7 questions (P=.01 and P<.001), whereas all questions showed a small level of increase. The two areas of significant knowledge improvement focused around the use of antibiotics for bacterial versus viral infections and ensuring the course of antibiotics is completed. Qualitative data showed that the e-Bug game “Body Busters” was the most popular, closely followed by “Doctor Doctor,” and “Microbe Mania” the least popular. Conclusions: This study shows that 2 of the e-Bug antibiotic educational games are valuable. “Body Busters” effectively increased antibiotic knowledge in children and had the greatest flow and enjoyment. “Doctor Doctor” also resulted in increased knowledge, but was less enjoyable. “Microbe Mania” had neither flow nor knowledge gain and therefore needs much modification and review. The results from the qualitative part of this study will be very important to inform future modifications and improvements to the e-Bug games.