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Objective: To determine whether a single-use stethoscope diaphragm barrier surface remains aseptic when placed on pathogen-contaminated stethoscopes. Methods: From May 31 to August 5, 2019, we tested 2 separate barriers using 3 different strains of 7 human pathogens, including extended-spectrum β-lactamase–producing Escherichia coli, methicillin-resistant Staphylococcus aureus, and vancomycin resistant Enterococcus faecium. Results: For all diaphragms with either of the 2 barriers tested, no growth was recorded for any of the pathogens. Stethoscopes with aseptic barriers remained sterile for up to 24 hours. These single-use barriers also provided aseptic surfaces when stethoscope diaphragms were inoculated with human specimens, including saliva, stool, urine, and sputum. Conclusion: Disposable aseptic diaphragm barriers may provide robust and efficient solutions to reduce transmission of pathogens via stethoscopes.