Prevalence of Mycoplasma species infections in chronic fatigue syndrome (CFS) has been extensively reported in the scientific literature. However, all previous reports highlighted the presence of Mycoplasmas in American patients. In this prospective study, the presence of Mycoplasma fermentans, M. penetrans, M. pneumoniae and M. hominis in the blood of 261 European CFS patients and 36 healthy volunteers was examined using forensic polymerase chain reaction. One hundred and seventy-nine (68.6%) patients were infected by at least one species of Mycoplasma, compared to two out of 36 (5.6%) in the control sample (P<0.001). Among Mycoplasma-infected patients, M. hominis was the most frequently observed infection (n=96; 36.8% of the overall sample), followed by M. pneumoniae and M. fermentans infections (equal frequencies; n=67; 25.7%). M. penetrans infections were not found. Multiple mycoplasmal infections were detected in 45 patients (17.2%). Compared to American CFS patients (M. pneumoniae>M. hominis>M. penetrans), a slightly different pattern of mycoplasmal infections was found in European CFS patients (M. hominis>M. pneumoniae, M. fermentansz.Gt;M. penetrans).