Clostridioides difficile poses an ongoing threat as a cause of gastrointestinal disease in human and animal populations. Traditionally considered a hospital disease, increases in community-associated human C. difficile infection (CDI) and growing evidence of transmission from animals and the environment necessitate a One-Health approach to the prevention and control of CDI. In horses, C. difficile is a major cause of diarrhoea and life-threatening colitis impacting both animal welfare and the financial integrity of equine industries. By determining prevalence, examining longitudinal data and using genomics, this study aimed to better understand the epidemiology of equine CDI in Western Australia (WA) and to provide insights into the relationships between horse, human and environmental strains of C. difficile. A total of 752 faecal samples from 387 domestic horses from stud farms, training centres, private properties and equine hospital settings in WA were cultured for C. difficile. Overall, C. difficile was isolated from 104 (30.9%) healthy horses and 19 (37.8%) horses with gastrointestinal symptoms - a rate significantly higher than previous studies within Australia. Of these 123 horses, 68 (55.3%) harboured toxigenic strains of C. difficile. Examination of equine hospital samples showed shedding of multiple strains in an individual horse in 13 (6.9%) subjects over the study period, suggesting both true colonisation and transient shedding existing simultaneously within this population. Both novel and known C. difficile ribotypes (RTs) were identified including prominent toxigenic human strains, 014/020 (n=15), RTs 012 (n=9), 087 (n=8), 002 (n=5) and 103 (n=2). Whole-genome analysis of 45 RT 012 strains of equine, human and environmental origin identified a genomic cluster of 10 isolates (0 to 62 SNPs difference, average 23) indicating recent shared ancestry. Evidence of possible clonal inter-species transmission or common source exposure (0-2 SNPs) was identified for a subgroup of three horse and one human isolates. This study highlights the need for a holistic approach to C. difficile surveillance and investigations to reduce CDI burden.