Hand hygiene promotion is considered as the cornerstone for healthcare-associated infection prevention. Over the past years, hand hygiene guidelines have been developed by different agencies at international, national and subnational levels. A comparison of these documents could help in understanding recommendations in different parts of the world and the methods used for their development. Guidelines were identified through search engines, electronic libraries, and personal contacts, and their content was analysed using an adapted version of a tool from the European DG XII-funded HARMONY project. Twenty-two guidelines were retrieved and 21 were evaluated. Documents varied in scope, approach, content and terminology. Some were primarily advisory directives, whereas others focused on the technical issues of why, when, and how to perform hand hygiene. The extent to which evidence was collected and assessed varied considerably and details were provided only in very few. Grading systems and definitions to indicate the strength of evidence and recommendations also differed. The intended outcome was to improve hand hygiene practices in healthcare, thus leading to a reduction of healthcare-associated infections and/or antimicrobial resistance. Although overall agreement on indications and procedures was noted, the range and depth of recommendations on best practices and implementation varied. Essential aspects such as compliance measurement and audits to assess guideline effectiveness were neglected in most documents. In conclusion, there is a need for a more consistent approach leading to recommendations based on a thorough evaluation of evidence and applicable worldwide. Aspects related to implementation and impact monitoring deserve greater attention.