Background: This study evaluated the frequency and types of hand hygiene practices among healthcare workers directed by the WHO multimodal hand hygiene improvement strategy, and investigated the effect of hand hygiene practice on methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) isolation and MRSA acquisition rate and colonization pressure. Methods: A quasi-experimental study was performed at a tertiary care university hospital with 850 beds from January to September 2012. We assessed the hospital hand hygiene program using the WHO hand hygiene self-assessment framework. The WHO multimodal strategy was used for healthcare workers with low indexes, and the subjects were reassessed. Results: Hand hygiene compliance increased significantly from a pre-intervention rate of 58.7% to 72.6% post-intervention. MRSA and VRE isolation rates decreased from 1.69 per 1000 patient days to 1.41 and from 0.17 to 0.11, respectively. In intensive care units (ICUs), hand hygiene compliance rate rose to 77.9%, with a total score of 4.16 points out of 5 being awarded for the hand hygiene method, which was higher than that for the other care units. The pre-intervention MRSA acquisition rate in the ICU decreased from 7.47% to 4.30% post-intervention. This was associated with a decrease in the MRSA colonization pressure over the intervention period (26.2% to 16.9%). Conclusion:The utilization of the WHO multimodal strategy for improvement of hand hygiene increased the hand hygiene compliance rate and was effective in predicting a decreased rate of cross-infection, MRSA acquisition, and colonization pressure. We conclude that the implementation of such improvement strategies is crucial to maintaining hygiene standards and reducing infection within healthcare facilities.