Stethoscopes have been suggested to be a possible vector of contact transmission. However, only a few studies have focused on the prevalence of contamination by multidrug-resistant (MDR) bacteria and effectiveness of disinfection training to reduce. This study is to investigate the burden of stethoscope contamination with nosocomial pathogens and multidrug-resistant (MDR) bacteria and to analyze habit changes in disinfection of stethoscopes among healthcare workers (HCWs) before and after education and training. We performed a prospective pre and post quasi-experimental study. A total of 100 HCWs (55 doctors and 45 nurses) were recruited. HCWs were surveyed on their disinfection behavior and stethoscopes were cultured by pressing the diaphragm directly onto a blood agar plate before and after education on disinfection. Pulsed-field gel electrophoresis was performed to determine the relatedness of carbapenem-resistant Enterobacteriaceae. Most of the stethoscopes were contaminated with microorganisms before and after the intervention (97.9% and 91.5%, respectively). The contamination rate of stethoscopes with nosocomial pathogens before and after education was 20.8% and 19.2%, respectively. Stethoscope disinfection habits improved (55.1% vs 31.0%; p<0.001), and the overall bacterial loads of contamination were reduced (median colony-forming units, 15 vs 10; p = 0.019) after the intervention. However, the contamination rate by nosocomial pathogens and MDR bacteria did not decrease significantly. A carbapenemase-producing Klebsiella pneumoniae isolates from a stethoscope was closely related to isolates from the patients admitted at the same ward where the stethoscope was used. Stethoscopes were contaminated with various nosocomial pathogens including MDR bacteria and might act as a vehicle of MDR bacteria. Continuous, consistent education and training should be provided to HCWs using multifaceted approach to reduce the nosocomial transmission via stethoscopes.