Objective: This study aimed to determine the expression of distinct matrix metalloproteinases, cytokines, and bone resorptive factors in temporomandibular joint osteoarthritis (TMJ-OA) patients and their association with joint pain, mouth opening, and subchondral bone degeneration. Materials and methods: Twelve patients affected with TMJ-OA (n = 5), disk displacement without reduction (DDWoR) (n = 3), or disk displacement with reduction (DDWR) (n = 4) were selected. Joint pain was quantified by using visual analog scale, mouth opening was quantified at the maximum pain-free aperture, and bone degeneration was quantified using joint imaging. Synovial fluid samples were collected and immediately processed for cell and synovial fluid recovering. From cells, the MMP-1, MMP-2, MMP-8, MMP-13, IL-6, IL-23, and TNF-α expression was quantified by qPCR. From synovial fluid, the RANKL and OPG levels were quantified by ELISA. Results: Higher levels of MMP-1, MMP-8, MMP-13, IL-6, IL-23, TNF-α, and RANKL/OPG ratio were detected in TMJ-OA compared with DDWoR and DDWR patients (p <.05). Joint pain significantly correlated with TNF-α levels (r =.975, p =.029). Besides, imaging signs of bone degeneration significantly correlated with RANKL/OPG ratio (r =.949, p =.042). Conversely, mouth opening did not correlate with any of the analyzed mediators. Conclusion: During TMJ-OA, a pathological response characterized by the overexpression of TNF-α and RANKL/OPG could be involved in joint pain and subchondral bone degeneration.