Introduction: Stethoscope is used to assess the health of patients but can also act as a potential source of disease transmission. The study was aimed to find out the contamination rate of stethoscopes, evaluate awareness and attitude of healthcare workers (HCWs) about stethoscope cleaning, and determine the efficacy of 70% alcohol as cleaning agent. Methodology: This hospital based cross-sectional study was conducted in a tertiary care hospital in October 2015 among healthcare workers. They were asked to fill a questionnaire followed by culturing the diaphragm and bell surfaces of their stethoscopes before and after cleaning with 70% isopropyl alcohol. Results: Out of 100 stethoscopes cultured, 56 were found to be contaminated at least with one microorganism. Acinetobacter cbc was the commonest contaminant followed by Klebsiella pneumoniae. Three out of twelve S. aureus strains showed methicillin resistance. Stethoscopes used in emergency areas were more contaminated when compared to wards and out-patient departments. Despite 100% awareness among HCWs, the importance of stethoscope cleaning is realized by only 70% who practice it regularly. Conclusion: Stethoscope is a potential vector for transmission of healthcare associated infections. Hence it is vital to clean it after each use to reduce the load of iatrogenic infections.