This study was designed to assess the knowledge, attitudes, and infection control practices among Nepalese health care workers (HCWs). The study comprised a questionnaire survey of 324 staff from acute care hospitals in Kathmandu, Nepal. A total of 158 doctors and 166 nurses participated, 27% of whom had received infection control training. Only 16%, 14%, and 0.3% of the respondents achieved maximum scores for knowledge, attitude, and practice items, respectively. Staff had good knowledge and positive attitude toward most aspects of infection control, although only half had heard of methicillin-resistant Staphylococcus aureus. Logistic regression revealed that profession, age, and having studied abroad significantly predicted markers of infection control knowledge, attitudes, and practice. This is the first survey of infection control practice among Nepalese HCWs and provides useful baseline data by professional group. There is ample opportunity for improvement in current practice, which should be recognized by hospital managers and Nepalese health authorities.