This study investigated whether the presence of Mycoplasma fermentans (M. fermentans) in the temporomandibular joint (TMJ) could be associated with the pathology of temporomandibular joint disorders (TMD). One hundred fifteen synovial fluid (SF) samples from patients with TMD were evaluated for the presence of DNA of M. fermentans by polymerase chain reaction (PCR) assay. Specific antibody against M. fermentans was also detected in the SF as well as sera by Western blot analysis. M. fermentans DNA was identified in 37.4% of the SF samples from the TMD patients. There was no difference between PCR-positive and -negative rate regarding sex and disease categories, e.g., internal derangement (ID) and osteoarthritis (OA). However, the prevalence of M. fermentans DNA in ID patients was higher in elderly patients (73.3%) than in younger patients (31.8%). Anti-M. fermentans immunoreactivities (IgG) specific for lipoproteins with various molecular sizes, 56 kilo-Dalton (kDa), 48 kDa, 38 kDa, and 29 kDa, were also identified in the SF. The immunoreactivity was also detected in the patients'sera. The reactivity patterns of the anti-M. fermentans antibodies were, however, different between the SF and the sera; reactivities to 48 kDa and 29 kDa lipoproteins were prominent in the former, while the reactivities to those of 56 kDa, 48 kDa, and 29 kDa were evidently increased in the latter. The presence of specific DNA and antibody for M. fermentans in the TMJ implies that M. fermentans could possibly induce joint specific immunoreaction, thus perpetuating the inflammatory reaction in the diseased TMJ.