The purposes of this study were to determine whether microorganisms can be isolated from the membranes of stethoscopes used by clinicians and nurses, and to analyse whether or not the degree of bacterial colonization could be reduced with different cleaning methods. We designed a transversal before-after study in which 122 stethoscopes were examined. Coagulase negative staphylococci (which are also potentially pathogenic microorganisms) were isolated together with 13 other potentially pathogenic microorganisms, including S. aureus, Acinetobacter sp. and Enterobacter agglomerans. The most effective antiseptic was propyl alcohol. Analysis of the cleaning habits of the Emergency Department (ED) staff, showed that 45% cleaned the stethoscope annually or never. The isolation of potentially pathogenic microorganisms suggests that the stethoscope must be considered as a potential vector of infection not only in the ED but also in other hospital wards and out-patient clinics.