This study investigated knowledge of and practices towards universal precautions among 540 health care workers and medical students in 2 university hospitals in Mazandaran Province, Islamic Republic of Iran. Only 65.8% and 90.0% staff in the 2 hospitals and 53.5% of medical students had heard about universal precautions. Overall, there was a low understanding of precautions, except concerning disposal of sharps, contact with vaginal fluid, use of mask and gown or cleaning spilled blood. Health workers had difficulty distinguishing between deep body fluids and body secretions that are not considered infectious. Good practices were reported regarding hand-washing, disposal of needles, and glove, mask and gown usage.