In the observational study emergency medical service (EMS) employees were observed during seven different hygiene measures by randomly selected EMS employees. Furthermore, a checklist was used to determine whether alcohol-based hand disinfectants, gloves, gowns and eye protection were available in the ambulance and in the emergency backpack. A total of 168 employees participated in the study: 123 nurses and 45 EMTs. 71 observations were carried out in 21 ambulances; 68 of these observations were analysed. The study showed that compliance with hand disinfection was 34% before and 72% after touching a patient. Gloves were correctly used in 69% of the situations, gowns in 91%. However, short-sleeved uniforms were only used in 28% of the observations. The checklists showed that hand disinfectants, gloves and gowns were available in all ambulances. Eye protection was available in approx. 90% of the ambulances. Hand disinfectants were available in almost every fifth backpack, gloves in every second. The hygiene compliance in EMS varies very much depending on the hygiene measure (only 3% compliant for all seven hygiene measures). The analysis of the checklists reveals that the access to the respective equipment was limited outside the ambulance, which may have a negative influence on the compliance.