Background: Disease outbreaks present a significant challenge to horse health and welfare and the economic stability of horse industries internationally. This is a particular concern in Ontario, Canada, where there have been frequent outbreaks of respiratory infectious diseases among horses. Despite these risks, there has been limited research on whether Ontario horse owners engage in biosecurity measures sufficient to mitigate risk of equine diseases, and whether current events such as the COVID-19 pandemic influence attitudes towards equine biosecurity practices., Objective: To explore Ontario horse owners' perceptions, attitudes and experiences relating to on-farm biosecurity during the COVID-19 pandemic., Study Design: Qualitative study using virtual semi-structured interviews., Methods: Participants (horse owners, frequent horse riders and part boarders) were recruited using social media snowball sampling where advertisements were shared by equine and veterinary organisations. Interviews were conducted virtually between June and September 2022 and were analysed using reflexive thematic analysis., Results: Three key themes relating to biosecurity perceptions among the 14 participants were identified. Participants relied on minimal preventative measures (such as vaccines) where perceived risk of disease was low, but implemented additional measures including quarantine and handwashing when perceived risk of disease was high. Participants' choice of biosecurity practices often mirrored those recommended by the barn manager. Moreover, participants felt that responsibility for biosecurity was not shared equally across horse owners, with more emphasis placed on those engaging in high-risk situations for disease spread. Despite experiencing biosecurity during the COVID-19 pandemic, horse owners were not consistently applying these practices to their horse care routines., Main Limitations: The perspectives reported here are from a small sample of horse owners and may not be generalisable to all populations., Conclusions: Our findings indicate that horse owners need improved access to and engagement with educational initiatives that emphasise the importance and purpose of all biosecurity measures., (© 2024 The Author(s). Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)