To assess knowledge and practices of healthcare workers (HCWs) in relation to bloodborne pathogens in a tertiary care hospital, western Saudi Arabia. Self-administered questionnaire was distributed assessing demographic characteristics, knowledge and practices of physicians, nurses and technicians on risks of exposure and prophylaxis against human immunodeficiency virus, hepatitis B virus and hepatitis C virus infections. A total of 466 participants (151; 32.4 % physicians and 315; 67.6 % nurses/technicians) completed the questionnaire. Almost two thirds of the physicians (60.9 %) and half of the nurses/technicians (47.6 %) had history of exposure to risks of bloodborne infection. Although both physicians and nurses/technicians showed acceptable level of knowledge about risks of bloodborne infections, modest proportion knew the correct actions including reporting following exposure. Behavioral-based in-service training interventions and strict policy should be implemented to promote compliance of HCWs to the protective measures against hazards of bloodborne infection.