Effective infection control practice requires knowledge of and adherence to contemporary infection control guidelines. Utilising a novel questionnaire tool, we evaluated knowledge of recently published guidelines on meticillin-resistant Staphylococcus aureus (MRSA) precautions in a number of relevant healthcare worker (HCW) populations. The questionnaire was developed from national UK MRSA practice guidelines and consisted of 10 'true or false' statements. The questionnaire was utilised to assess knowledge in 293 participants from HCW and control populations. The participants included 188 doctors attending the British Medical Association's Annual Representatives Meeting, 52 trainee surgeons attending the Association of Surgeons in Training annual conference, 30 members of a non-clinical control population and 23 infection control nurses (ICNs). The mean (SD) score for knowledge levels obtained from doctors was 6.6 (1.68), for non-clinical control population was 4.7 (1.8) and for ICNs, 8.4 (1.12). There were significant differences in knowledge levels between different population groups (P<0.001), UK employment region of the participant (P=0.01) and the doctors' medical specialty (P=0.02). Career seniority and gender of the participant were not significantly associated with differences in levels of knowledge. This questionnaire study evaluates a novel discriminatory questionnaire tool which differentiates knowledge levels of MRSA practice guidelines among a non-clinical population, HCWs and specialist infection control staff, thus providing a means for the rapid assessment of MRSA educational interventions. We identify demographics within HCW target populations which are associated with low levels of such knowledge. Consideration towards revising current HCW educational programmes to improve knowledge and best practice in MRSA prevention is required.