Osteoarthritis (OA) causes persistent joint deterioration, severe morbidity, and reduced mobility in both humans and horses. Extracellular vesicles (EVs) are key players in cell-to-cell communication and are thus ideal for biomarker discovery. Here, we used a unique multi-omics strategy to find possible EV biomarkers from the synovial fluid of healthy horses compared to naturally occurring OA and advanced OA. We discovered that SF-EVs’ proteome and phospholipidome are correlated. Furthermore, we found that the quantity of EVs present in SF was unaffected by OA. We discovered crucial signalling pathways involving integrins (p=2.72x10-24), the actin cytoskeleton (p=3.88x10-32), Rho family GTPases (p=1.24x10-19), and the clathrin-mediated endocytosis pathway (p=1.30x10-24). Specifically, the advanced OA group’s immunological response (macrophage binding and interaction) was upregulated. These findings are suggestive that the membrane and protein cargo of SF-EVs are altered in response to OA pathology and may be used as biomarkers of disease. Consequently, SF-EVs could have the potential to be employed in the future to help with OA stratification and prognosis. In addition, this study provides a framework for future research using a larger cohort.