Mycoplasma pneumoniae (Mp) is a frequent cause of Community Acquired Pneumoniae (CAP). The etiological role of Mp is usually suspected using serological assays, but the detection of specific anti-Mp antibodies becomes possible only 1-2 weeks after the primary infection. On the contrary, direct diagnosis using real-time PCR allows an efficient detection of Mp DNA in all the phases of the infection and particularly during early serum negative periods. In this study, we developed a novel Scorpion-probe real-time PCR-based assay. The probe's uni-molecular structure offers thermodynamic advantages owing to its kinetic reaction, providing faster performances compared to a TaqMan-based assay, but maintaining the same sensitivity and specificity. The Scorpion-based assay was employed on 388 clinical samples and compared with conventional qualitative PCR and serological tests. It was found more sensitive because it also allowed the detection of Mp in specimens found negative using classic qualitative PCR, but displaying seropositivity or a later seroconversion.