Background: There is limited research on the importance of implementation when evaluating the effect of hand hygiene interventions in school settings in developed countries. The aim of this study was to examine the association between an implementation index and the effect of the intervention. The Hi Five Intervention was evaluated in a 3-armed cluster randomized controlled trial involving 43 randomly selected Danish schools. Methods: Analyses investigating the association between implementation of the Hi Five Intervention and infectious illness days, infectious illness episodes, illness-related absenteeism, and hand hygiene were carried out in a multilevel model (school, class, and child). Results: The level of implementation was associated with hand hygiene and potentially associated with number of infectious illness days and infectious illness episodes among children. This association was not found for illness-related absenteeism. Conclusions: Classes that succeeded in achieving a high level of implementation of the Hi Five Intervention had a lower number of infectious illness days and infectious illness episodes, suggesting that the Hi Five Intervention, if implemented adequately, may be relevant as a tool to decrease infectious illness in a Danish school setting.