Bio-medical waste has a higher potential of infection and injury to the healthcare worker, patient and the surrounding community. Awareness programmes on their proper handling and management to healthcare workers can prevent the spread of infectious diseases and epidemics. This study was conducted in a tertiary care hospital to assess the impact of training, audits and education/implementations from 2009 to 2012 on awareness and practice of biomedical waste segregation. Our study reveals focused training, strict supervision, daily surveillance, audits inspections, involvement of hospital administrators and regular appraisals are essential to optimise the segregation of biomedical waste.