Hand hygiene, cleaning and disinfection are pre-requirements for hygiene management in hospital settings and the food industry. In order to facilitate risk management, different contamination scenarios and interventions need to be evaluated. In the present study data on transfer rates and reductions of Staphylococcus aureus were provided in an experimental set-up using artificial skin. Using this methodology, test persons were not exposed with pathogenic bacteria. An exposure assessment model was developed and applied to evaluate different contamination routes and hygiene interventions. The transfer rates of S. aureus from inoculated VITRO-SKIN® to fomites were calculated from blotting series. The VITRO-SKIN® was more prone to spread bacteria than fomites. When different surfaces were cleaned, the reduction of S. aureus varied between <1 and 7 log CFU. It could not be concluded that a certain coupon material, cleaning agent, cleaning wipe, soiling or humidity consistently resulted in a high or low reduction of S. aureus. The reduction of S. aureus and E. coli during hand washing was evaluated on artificial skin, VITRO-SKIN®. The reduction of E. coli on VITRO-SKIN® was similar to the log reduction obtained when washing human hands. The S. aureus count on a human hand was both calculated in different scenarios describing different contamination routes starting from a contaminated hand using the exposure assessment model, and measured on an experimental setup using VITRO-SKIN® for validation. A linear relationship was obtained between the analysed level of S. aureus and the calculated level. However, the calculated levels of S. aureus on the VITRO-SKIN® in the scenarios were 1–1.5 log lower than the analysed level. One of the scenarios was used to study the effect of interventions like hand washing and cleaning of surfaces.