Q fever (caused by Coxiella burnetii) is a serious zoonotic disease that occurs almost worldwide. Occupational contact with animals increases the risk of exposure, and Q fever vaccination is recommended for veterinary workers in Australia. This study aimed to investigate C. burnetii seroprevalence among unvaccinated veterinary workers in Australia and determine factors associated with a positive serological result. During 2014 and 2015, convenience sampling at veterinary conferences and workplace vaccination clinics was undertaken. Participants completed a questionnaire and provided a blood sample for C. burnetii serology. Participants were predominantly veterinarians (77%), but veterinary support staff, animal scientists, and administration workers also participated. Blood samples (n = 192) were analysed by an immunofluorescence assay and considered positive where the phase I or phase II IgG titre was ≥1/50. Seroprevalence was 19% (36/192; 95% CI 14%–25%). A positive serological result was significantly associated with (a) working in outer regional/remote areas (odds ratio [OR] 6.2; 95% CI 1.9–20.8; reference = major cities; p =.009) and (b) having spent more than 50% of total career working with ruminants (OR 4.8; 95% CI 1.7–13.5; reference = <15% of career; p =.025). These findings confirm an increased risk of exposure to C. burnetii compared to the general population, providing new evidence to support Q fever vaccination of veterinary workers in Australia.