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Hand hygiene is a cornerstone of infection prevention. However, few data are available for school children on their knowledge of infectious diseases and their prevention. The aim of the study was to develop and apply a standardized questionnaire for children when visiting primary schools to survey their knowledge about infectious diseases, pathogen transmission and prevention measures. Enrolling thirteen German primary schools, 493 questionnaires for grade three primary school children were included for further analyses, comprising 257 (52.1%) girls and 236 (47.9%) boys with an age range of 8–11 years. Out of 489 children, 91.2% participants indicated that they knew about human-to-human transmissible diseases. Of these, 445 children responded in detail, most frequently mentioning respiratory and gastrointestinal diseases, followed by childhood diseases. Addressing putative hygiene awareness-influencing factors, it was worrisome that more than 40.0% of the children avoided visiting the sanitary facilities at school. Most of the children (82.9%) noted that they did not like to use the sanitary facilities at school because of their uncleanliness and the poor hygienic behavior of their classmates. In conclusion, basic infection awareness exists already in primary school age children. Ideas about the origin and prevention of infections are retrievable, however, this knowledge is not always accurate and adequately contextualized. Since the condition of sanitary facilities has a strong influence on usage behavior, the child’s perspective should be given more consideration in the design and maintenance of sanitary facilities.