the objective of this study was to evaluate knowledge, practice and attitude of nurses, auxiliary nurses and midwifery staff towards standard isolation precautions. In a cross-sectional survey 200 nurses, auxiliary nurses and midwifery staff were recruited to complete a questionnaire about their knowledge, attitude and practice towards standard isolation precautions in a teaching hospital in Yazd, Iran. the data were collected and analyzed using SPSS 14. the initial sample consisted of 230 participants, of whom 200 (87%) responded to the distributed questionnaires. Knowledge and attitudes were moderate but their practice was relatively poor. Mean score of knowledge, attitude and practice towards standard isolation precautions were 11.9 (Maximum = 16.5), 27 (Maximum = 50), 4.6 (Maximum = 9), respectively. Practice scores of women were significantly higher than men (P = 0.028). The score of Knowledge increased significantly with age (P = 0.004). A positive linear correlation was observed between knowledge and practice (P =0.01, correlation coefficient = 0.173). this study revealed that there were serious defects in knowledge of health care workers regarding hand hygiene, disinfecting endoscopes, avoiding recapping needles and respiratory isolation precautions.