Objective Soluble mediators in synovial fluid are acknowledged as key players in the pathophysiology of osteoarthritis (OA). However, a wide-spectrum screening of such mediators in synovial fluid is currently lacking. In this study, the levels of 47 mediators in the synovial fluid of control donors and osteoarthritic (OA) patients were compared. Materials & Methods Synovial fluid was collected from control donors (n=16) and end-stage knee OA patients (n=18) and analysed for 47 cytokines, chemokines and growth factors using several multiplex ELISAs. A Mann-Whitney U test was used to determine differences between OA and control controls. A principal component analysis (PCA) was performed to cluster the 47 mediators. Results The majority of the mediators could be detected in both control and OA synovial fluid. IL-6, IP-10, MDC, PDGF-AA and RANTES levels were found to be higher in OA compared to control synovial fluid (p<0.001). Leptin, IL-13, MIP-1β, sCD40L levels were higher and eotaxin and G-CSF levels were lower in OA synovial fluid than in control synovial fluid, albeit borderline significant (p<0.05). The PCA enabled identification of 6 clusters of mediators, which explained 76% of the variance. Conclusions The current study provides the first extensive profile of cytokines, chemokines and growth factors present in control and OA synovial fluid. Increased levels of mediators such as MDC and IL-6 imply involvement of inflammatory processes and might be associated with the influx of inflammatory cells in OA synovial tissue. Moreover, the performed cluster analysis indicated multiple clusters, which could indicate different pathophysiological pathways in the joint.