The objective of this study was to analyze knowledge on hand hygiene among staff workers in the Andalusian Public Health System (Spain). This was a cross-sectional study with surveys (2011) using the Hand Hygiene Knowledge Assessment Questionnaire for Healthcare Workers with the latent class analysis technique. The average number of questions answered correctly was 17.51±3.68. Questions with lower percentages of correct responses were those on hand hygiene for prevention of microorganism transmission to patients and those on hand- rubbing versus hand-washing. We obtained a model with 7 latent classes. Workers with lower knowledge tended to be younger, males, and non-healthcare workers. Having received previous training did not necessarily ensure excellent knowledge. The study concludes that hand hygiene training programs need to be revised in order to improve knowledge on conceptual characteristics involved in the transmission of microorganisms via the hands.