Hendra virus is a zoonotic paramyxovirus, which causes severe respiratory and neurological disease in horses and humans. Since 2012, the Hendra virus sub-unit G vaccine has been available for horse vaccination in Australia. Uptake of the vaccine has been limited and spill-over events of Hendra virus infection in horses continue to occur. We conducted an online, questionnaire-based cross-sectional study of 376 horse owners belonging to a variety of different equestrian clubs in Queensland, Australia, to identify risk factors for non-vaccination against Hendra virus. A total of 43.1% (N = 162) of horse owners indicated that they currently did not vaccinate against Hendra virus infection, while 56.9% (N = 214) currently vaccinated against Hendra virus infection. A total of 52 risk factors were evaluated relating to equestrian activities, horse management, perceived risk and severity of horse and human infection with Hendra virus, side effects of Hendra vaccination, other vaccinations conducted by horse owners and horse owners' attitudes towards veterinarians. The final multivariable logistics regression model identified the following risk factors associated with increased odds of non-vaccination against Hendra virus: 1) perceived low risk (compared to high) of Hendra virus infection to horses (considering the horse owners' location and management practices) or horse owners were unsure about the risk of infection, 2) perceived moderate severity (compared to very severe or severe) of Hendra virus infection in humans, 3) horse owners non-vaccination of their pets, 4) horse owners non-vaccination against strangles disease in horses, 5) handling of more than three horses per week (compared to one horse only) and 6) perceived attitude that veterinarians had a high motivation of making money from Hendra virus vaccination (compared to veterinarians having a low motivation of making money from Hendra virus vaccination). Horse owners were more likely to vaccinate against Hendra virus if horses were used for dressage, show jumping or eventing. The study also identified horse owners' concerns about side-effects and about the lack of evidence on vaccine efficacy.