Aims: To assess the knowledge of Biomedical waste (BMW) categories, colour coding, transport, storage & disposal of Biomedical waste among the healthcare workers. Study Design: Cross sectional. Place and Duration of Study: Multi speciality Hospital, Pune, over the duration of 1 month. Methodology: A predesigned questionnaire containing closed-ended questions was used to conduct this cross sectional study on HCWs. The data related to awareness & knowledge about various aspects of Biomedical waste amongst the Healthcare workers was collected. Results: Out of total 100 Healthcare workers (HCWs), 40 doctors were correctly knowing all the categories of Biomedical waste. 45 doctors & 40 nursing staff (total 85 out of 100 HCWs) could answer correctly the questions on colour coding of BMW. Only 30 doctors & 21 nursing staff could answer correctly about BMW transport. 25 doctors &18 nurses could answer correctly the questions related to BMW storage & disposal. Conclusion: The vigorous & repeated training & evaluation is needed to bridge the observed knowledge gaps amongst the HCWs.