The analysis of synovial fluid is crucial for evaluating joint pathologies, providing specific diagnoses, and guiding treatment. However, consensus on synovial fluid analysis in the temporomandibular joint (TMJ) is lacking. This study evaluates the relationship between biochemical markers in TMJ synovial fluid and the severity of temporomandibular dysfunction. Clinical data from 379 arthroscopies performed between 2008 and 2022 were reviewed retrospectively. Outcome assessments were based on the concentration of protein, glucose, sodium, potassium, and chloride in the synovial fluid of patients with internal derangement. Independent variables included Wilkes stages, arthroscopic findings, and the color of the fluid. Protein concentration was significantly associated with synovitis, chondromalacia, and roofing. Higher glucose levels correlated with increased synovitis, decreased roofing, and advanced Wilkes stages. Elevated chloride concentrations were linked to worsening chondromalacia and roofing. Sodium concentration showed a significant association with disc displacement, while elevated potassium levels were connected to advanced Wilkes stages and synovitis. Notably, higher levels of protein, glucose, chloride, sodium, and potassium were associated with hemorrhagic synovial fluid. This study highlights that analyzing these markers can promote early detection and management of temporomandibular disorders. Further research is needed to validate these findings and explore additional clinical applications.