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ABSTRACT Hand hygiene is one of the most important infection prevention and control strategies to reduce pathogen transfer in healthcare settings. While there are dedicated evidence-based hand hygiene interventions and protocols to support human healthcare providers, there are no comparable resources to support veterinary care. A pilot trial for the implementation of a hand hygiene education and compliance program was conducted in six heterogenous small animal veterinary practices. Hand hygiene compliance was evaluated using a standardised and validated program, based on the World Health Organization’s 5 Moments for Hand Hygiene program. Veterinary Nurse Champions undertook dedicated hand hygiene training and were supported by the first author throughout the pilot trial. Hand hygiene compliance was low (14%) pre-trial, improved to 46% after the six-week trial period and remained at 35% six months after conclusion of the trial. Compliance for all five Moments improved compared to the pre-trial period in the immediate post-trial period. Of the five Moments, there was a statistically significant increase in compliance with Moments 3 (after a procedure/body fluid risk), 4 (after touching a patient) and 5 (after touching a patient’s surroundings) in the immediate post-trial period, however, by 6 months post-trial, only Moment 5 showed a significant improvement. Barriers to improvement included poor availability of hand hygiene products, lack of time due to clinical workload requirements, lack of awareness, and an ingrained workplace culture.