Stethoscopes are potential vector for health care associated infections worldwide. Ethyl alcohol (EA), isopropyl alcohol (IPA) and chlorhexidine (CH) are widely used for disinfecting stethoscope, however, comparative analysis of these disinfectants are scarce. Hence this paper aims to compare the reduction in bioburden with respect to stethoscope decontamination. A randomized, double-blind study was undertaken from various categories of health care workers (HCW) working in various departments. Each participant was asked to fill a questionnaire and diaphragms of their stethoscopes were imprinted on blood agar and sabourauds dextrose agar before and after disinfection with either 65%-EA, 70%-IPA or 1%-CH. The culture plates were incubated aerobically and the growth was further identified. 384 HCW participated in the study. 168 participants never cleaned their stethoscope, out of which 147 respondents (91.6%) comprised of students. EA (47%) and IPA (23%) were the two major types of alcohols used as disinfecting agents. 280 samples showed growth among which 51.8% were potent pathogens. Before disinfection, Staphylococcus aureus (27%) was the predominant pathogen, followed by Klebsiella (6.8%), Enterococcus and Candida respectively. Among the disinfectants used, maximum antimicrobial activity was exhibited by IPA (92.5%) compared to EA (82.5%) and CH (77.8%). Huge lacunae regarding knowledge, attitude and practice of stethoscope maintenance was found among student community. The microbial growth varies at various department. Disinfecting stethoscope with 70%-IPA for just 30 seconds is equally efficient compared to 1 minute and IPA was superior to EA and CH for decontaminating stethoscope. Stethoscopes are potential vector for health care associated infections worldwide. Ethyl alcohol (EA), isopropyl alcohol (IPA) and chlorhexidine (CH) are widely used for disinfecting stethoscope, however, comparative analysis of these disinfectants are scarce. Hence this paper aims to compare the reduction in bioburden with respect to stethoscope decontamination. A randomized, double-blind study was undertaken from various categories of health care workers (HCW) working in various departments. Each participant was asked to fill a questionnaire and diaphragms of their stethoscopes were imprinted on blood agar and sabourauds dextrose agar before and after disinfection with either 65%-EA, 70%-IPA or 1%-CH. The culture plates were incubated aerobically and the growth was further identified. 384 HCW participated in the study. 168 participants never cleaned their stethoscope, out of which 147 respondents (91.6%) comprised of students. EA (47%) and IPA (23%) were the two major types of alcohols used as disinfecting agents. 280 samples showed growth among which 51.8% were potent pathogens. Before disinfection, Staphylococcus aureus (27%) was the predominant pathogen, followed by Klebsiella (6.8%), Enterococcus and Candida respectively. Among the disinfectants used, maximum antimicrobial activity was exhibited by IPA (92.5%) compared to EA (82.5%) and CH (77.8%). Huge lacunae regarding knowledge, attitude and practice of stethoscope maintenance was found among student community. The microbial growth varies at various department. Disinfecting stethoscope with 70%-IPA for just 30 seconds is equally efficient compared to 1 minute and IPA was superior to EA and CH for decontaminating stethoscope.