Background. Healthcare workers have an increased risk of latent tuberculosis infection (LTBI), but previous studies suggested that they might be reluctant to accept preventive tuberculosis (TB) treatment. We aimed to examine doctors' and nurses' experience of TB screening and to explore their attitudes towards preventive TB treatment. Methods. We conducted a survey among randomly selected healthcare workers at a tertiary hospital in Sydney, Australia, using a paper-based questionnaire. Results. A total of 1,304 questionnaires were distributed and 311 (24%) responses were received. The majority of hospital staffsupported preventive TB treatment in health care workers with evidence of latent TB infection (LTBI) in general (74%, 164/223) and for them personally (81%, 198/244) while 80 and 53 healthcare workers respectively had no opinion on the topic. Staffworking in respiratory medicine were significantly less likely to support preventive TB treatment in health care workers in general or for them personally if they would have evidence of LTBI compared to other specialties (p = 0:001). Only 13% (14/106) of respondents with evidence of LTBI indicated that they had been offered preventive TB treatment. Twenty-one percent (64/306) of respondents indicated that they did not know the difference between active and latent TB. Among staffwho had undergone testing for LTBI, only 33% (75/230) felt adequately informed about the meaning of their test results. Discussion. Hospital staffin general had positive attitudes towards preventive TB treatment, but actual treatment rates were low and perceived knowledge about LTBI was insufficient among a significant proportion of staff. The gap between high support for preventive TB treatment among staffand low treatment rates needs to be addressed. Better education on the concept of LTBI and the meaning of screening test results is required.