Introduction: Hand hygiene is the most effective measure to prevent hospital-acquired infections, particularly in high-risk areas such as the Pediatric Intensive Care Unit (PICU). Adherence to the World Health Organization (WHO) “Five Moments for Hand Hygiene” is essential to reduce cross-transmission. Objectives: To assess hand hygiene compliance according to WHO guidelines, compare practices among doctors, nurses, and allied health professionals (AHPs), and identify barriers to adherence. Methods: A prospective observational audit was conducted in the PICU at National Hospital Galle using the WHO Hand Hygiene Observation Form. Fifty hand hygiene opportunities were observed across staff categories. Results: Overall compliance was 62%. Compliance was highest before aseptic procedures (100%) and after body fluid exposure (83.3%). Nurses showed the highest compliance (83.3%), followed by AHPs (75%) and doctors (58.8%). Noncompliance was common during busy periods. Conclusion: Hand hygiene compliance remains suboptimal, highlighting the need for regular training, reinforcement, and repeat audits.