Orthopedic prosthesis infection must be medically managed after appropriate microbiological documentation. While bacteria and fungi are acknowledged to be causative opportunistic pathogens in this situation, the potential role of methanogens in orthopedic prosthesis infections is still unknown. In a retrospective study, a total of 100 joint and bone samples collected from 25 patients were screened by specific PCR assays for the detection of methanogens. PCR-positive samples were observed by autofluorescence, electron microscopy and tentatively cultured under specific culture conditions. Methanogens were detected by quantitative PCR in 4/100 samples, in the presence of negative controls. Sequencing identified Methanobrevibacter oralis in two cases, Methanobrevibacter smithii in one case and Methanobrevibacter wolinii in one case. Microscopic methods confirmed molecular findings and bacterial culture yielded two strains of Staphylococcus aureus, one strain of Staphylococcus epidermidis and one strain of Proteus mirabilis. These unprecedented data highlight the presence of methanogens in joint and bone samples of patients also diagnosed with bacterial orthopedic prosthesis infection, questioning the role of methanogens as additional opportunistic co-pathogens in this situation.