In recent years, outbreaks of exotic as well as newly emerging infectious diseases have highlighted the importance of biosecurity for the Australian horse industry. As the first potentially fatal zoonosis transmissible from horses to humans in Australia, Hendra virus has emphasised the need to incorporate sound hygiene and general biosecurity practices into day-to-day horse management. Recommended measures are widely publicised, but implementation is at the discretion of the individual owner. This cross-sectional study aimed to determine current levels of biosecurity of horse owners and to identify factors influencing the uptake of practices utilising data from an online survey. Level of biosecurity (low, medium, high), as determined by horse owners’ responses to a set of questions on the frequency of various biosecurity practices performed around healthy (9 items) and sick horses (10 items), was used as a composite outcome variable in ordinal logistic regression analyses. The majority of horse owners surveyed were female (90%), from the states of Queensland (45%) or New South Wales (37%), and were involved in either mainly competitive/equestrian sports (37%) or recreational horse activities (35%). Seventy-five percent of owners indicated that they follow at least one-third of the recommended practices regularly when handling their horses, resulting in medium to high levels of biosecurity. Main factors associated with a higher level of biosecurity were high self-rated standard of biosecurity, access to personal protective equipment, absence of flying foxes in the local area, a good sense of control over Hendra virus risk, likelihood of discussing a sick horse with a veterinarian and likelihood of suspecting Hendra virus in a sick horse. Comparison of the outcome variable with the self-rated standard of biosecurity showed that over- as well as underestimation occurred. This highlights the need for continuous communication and education to enhance awareness and understanding of what biosecurity is and how it aligns with good horsemanship. Overall, strengthened biosecurity practices will help to improve animal as well as human health and increase preparedness for future disease outbreaks.