Background: Infectious diseases pose significant challenges in pediatric daycare centers due to high contact rates among children. Hand hygiene is a critical measure in preventing illness transmission. Methods: This cluster randomized trial involved 800 children across 20 daycare centers, divided into handwashing, hand sanitizers, educational program, and control groups. The study assessed the interventions' effectiveness in improving hand hygiene compliance, reducing microbial loads, and decreasing illness episodes and absenteeism. Results: Post-intervention, hand hygiene compliance significantly increased in the handwashing (85 ± 4.3%, p<0.01), hand sanitizers (82 ± 4.7%, p<0.01), and educational program groups (83.5 ± 4.2%, p<0.01). The educational program most effectively reduced illness episodes (from 59% to 35%, p<0.01) and absenteeism (from 29% to 13%, p<0.01). Microbial load reductions were highest in the hand sanitizers group (hands: 55%, surfaces: 53%, p<0.01). Factors influencing compliance included age, gender, and the presence of visual reminders. Conclusion: While all interventions improved hand hygiene and reduced illness transmission, the educational program demonstrated the greatest efficacy. Incorporating visual reminders and tailoring strategies to demographic specifics can further optimize hand hygiene practices in pediatric settings.