The aim of this study was to explore the diagnostic value of matrix metalloproteinase (MMP)-2 and -9, and cyclooxygenase-2 (COX-2) enzymes in the synovial fluid of horses with different forms of arthritis. Thirty-two horses were involved in this study and based on the clinical, radiographic, and synovial fluid examinations, the horses were divided into three groups: control (group I; n = 12), septic arthritis (group II; n = 5), and aseptic arthritis (group III; n = 15). After routine analysis, synovial fluid was used for assessment of MMP-2 and MMP–9 activities by gelatin zymography, and COX-2 relative gene expression by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Synovial fluid gelatin zymography showed significant gelatinolytic activity for MMP-9 in group II. COX-2 exhibited a 50-fold expression in group II and a 4.5-fold expression in group III compared to group I. In conclusion, the results confirm that MMP-9 and COX-2 detection offers an important diagnostic potential for septic and aseptic arthritis in horses.