The aetiological diagnosis of pneumonia depends largely on culture-, antigen- or PCR-based tests. Atypical agents of pneumonia include Coxiella burnetii, Chlamydophila pneumoniae, Chlamydia psittaci, Legionella pneumophila, Francisella tularensis and Mycoplasma pneumoniae. In these cases, serological tests are commonly used for diagnosis. All of the above species were comparatively screened for by using the InoDiag multiplexed automatic immunofluorescence assay and established reference techniques. The InoDiag assay required 5 microL of serum, took 76 min per serum sample, and required an incubator, a fluorescence reader and interpretation software. In total, 248 single sera from patients were tested, for the diagnosis of pneumonia, and the results obtained with selected serum samples were compared with results obtained with the reference method. It was shown that, for the detection of Coxiella burnetii IgM, the automated assay had a sensitivity and specificity of 100%. For the detection of M. pneumoniae IgM, sensitivity was 100% and specificity was 98%. For the detection of Chlamydophila pneumoniae and Chlamydia psittaci IgG, sensitivity was 81% and specificity was 94%. For the detection of L. pneumoniae IgG, sensitivity was 63% and specificity was 98%. For the detection of F. tularensis IgG and IgM, sensitivity was 100% for both, and specificity was 95% and 100%, respectively. The performance of this serological assay was comparable to that of other assays reported in the literature. This preliminary study shows that the automatic InoDiag assay opens the way to immunofluorescence assay standardization.