Anaesthetists are a group of doctors who are at a higher occupational risk of contracting blood borne infections as they perform many invasive procedures. Practice of Universal Precautions (UP) is vital in reducing the risk of transmission. The objective of this study was to assess the knowledge and compliance among anaesthetists of the National Hospital of Sri Lanka (NHSL) on UP and related issues. Information was collected from 97 anaesthetists at the NHSL using a structured questionnaire. Knowledge and practice of UP was assessed separately through specific questions. The average percentage mark of the 97 respondents on their knowledge on UP was 77.43% with a standard deviation of 13.84. Approximately, 68% of the respondents obtained above-average marks. With regard to practise of UP, 61% of the respondents had their total coded mark within the range from 14 to 16 out of the possible maximum of 21. There was no significant correlation between knowledge and practice. Only 15% of the population wore gloves during all procedures at OT, and 46% has stated that it is comfortable to work without gloves as the reason for not wearing. Based on these results, it is concluded that there is appreciable room for improvement of knowledge and practice of UP among anaesthetists. The study further revealed that knowledge and practice of UP is not related to gender or experience, which indicated that it is probably a personal trait of each respondent, depending on the discipline and application of each individual.