Introduction: Stethoscopes serve as a potential source for pathogenic bacteria in the transmission of hospital acquired infections. Routine disinfection of stethoscopes could reduce the potential for transmission by reducing the burden of contamination. Despite these facts, a few health care workers disinfect their stethoscopes regularly. Aim: To study the diversity of pathogenic organisms on the diaphragm of stethoscopes and the efficacy of various disinfectants will be compared; and the knowledge and practices in disinfection of stethoscopes among health care workers will be assessed. Materials & Methods: This is a cross sectional study involving a total of 132 health care workers including doctors, nurses and interns. Three swabs were collected from the same individual’s stethoscopes. First swab collected before disinfection, second swab after disinfection (soap and water, 70% isopropyl alcohol, Savlon or surgical spirit) and the third swab after examining a patient. Questionnaire was given to them to evaluate their practices regarding cleaning of their stethoscopes. Results: 94(71.21%) samples taken before disinfection were contaminated with mixed microbial flora, including 29(30.85%) MRSA. After examining patients using disinfected stethoscopes, 103(78%) samples revealed growth of different organisms like Methicillin Resistant Staphylococcus aureus-MRSA, Methicillin sensitive Staphylococcus aureus-MSSA, Gram negative bacilli and other normal skin flora. 112(84.85%) participants accepted that stethoscopes could transmit infectious diseases and they must be cleaned regularly. But in practice, only 72(54.55%) are cleaning it and most of them were using surgical spirit. Out of the four disinfectants tested, Surgical spirit and 70% Isopropyl alcohol were found to be effective. Conclusion: Despite having knowledge about the significance of cleaning the stethoscopes, lesser percentage of Healthcare workers follow it in practice. There is a high transmissibility of human skin flora along with pathogens like MRSA among patients through stethoscopes. Thus, stethoscopes act as a potential vector in the spread of nosocomial infections. Hence it is mandatory to clean and disinfect the stethoscopes after each patient examination.