School-aged children are vulnerable to infectious diseases due to their developing immune systems and frequent social interactions. The COVID-19 pandemic underscored the importance of non-pharmaceutical interventions, like hand hygiene (HH). This study evaluated the changes achieved through a school-based intervention to Guatemalan primary school students’ HH knowledge, attitudes, and self-reported practices while collecting teacher feedback to inform future efforts. The intervention included handwashing festivals, environmental nudges, and the regular delivery of soap and alcohol-based hand rub (ABHR). Knowledge, attitudes, and practices (KAP) surveys were conducted pre- and post-intervention with 109 and 144 students, respectively. Six teachers participated in interviews to provide perspectives. Significant improvements were observed in students’ knowledge of HH’s role in preventing disease (pre: 84.4%; post: 96.5; p < 0.01) and recognition of critical moments (pre: 84.4%; post: 92.4%; p < 0.05). Self-reported practices also improved, with more students reporting washing their hands for 20 s or more (pre: 68.8%; post: 79.9%; p < 0.05). Fewer students reported liking ABHR after the intervention (pre: 89%; post: 78.5%; p < 0.05). Teachers reported increased HH practices and provided feedback to enhance interventions. These findings highlight the effectiveness of school-based interventions and emphasize the importance of addressing knowledge gaps and incorporating teacher insights for sustained public health benefits.