Invasive aspergillosis is an opportunistic infection caused primarily by Aspergillus fumigatus. However, other common fungal pathogens belonging to section Fumigati are often misidentified as A. fumigatus. Thus, we have developed a multiplex real-time PCR (qPCR) assay with primers and specific TaqMan probes based on internal transcribed spacer regions or benA gene to discriminate, in less than 3 h, species of section Fumigati and, specifically, A. fumigatus. The multiplex qPCR showed a limit of detection of 20 and 50 fg of DNA for section Fumigati and A. fumigatus, respectively. Moreover, it enabled detection of a single germinated conidia. The inclusion of some PCR facilitators together with the dilution of samples makes it possible to completely avoid PCR inhibitions in all bronchoalveolar lavage (BAL) samples assayed. This technique may be a useful complementary tool in the diagnosis of invasive pulmonary aspergillosis caused by A. fumigatus using BAL fluid.