The serological diagnosis of blood culture-negative endocarditis due to Coxiella burnetii, Bartonella spp., Brucella melitensis and Legionella pneumophila is based on a manual immunofluorescence assay (IFA), which is taken to be the reference method. The automated IFA InoDiag multiplexed antigenic microarray, which includes a slide with all the above bacteria and four internal controls, an incubator, a fluorescent reader and software with an algorithm of interpretation for infectious endocarditis (IE) was evaluated. A single serum dilution at 1/128 was used. Eleven patients with Bartonella spp. IE and ten with C. burnetii IE, diagnosed using the modified Duke criteria, as well as one patient with B. melitensis infection and three patients with L. pneumophila IE were tested. In total, 236 sera were used as negative controls, with the reference method. The results of IgG detection were: C. burnetii phase I, 'sensitivity (Se) = 88% and specificity (Sp) = 99%', and C. burnetii phase II, Se = 88% and Sp = 99%; for Bartonella henselae, Se = 100% and Sp = 100%; for Bartonella quintana, Se = 78% and Sp = 96%; for B. melitensis, Se = 100% and Sp = 99%; and for L. pneumophila, Se = 100% and Sp = 99%. With the algorithm interpretation, the negative and positive predictive values of the test 'were 100% for the diagnosis of IE caused by the four bacteria tested. These results were confirmed by two other assays, one using triplicate testing and one blind testing performed by another centre. This multiplexed test is therefore a valuable tool for the rapid diagnosis of blood-culture negative IE.