Enzyme-linked immunosorbent assays (ELISAs) used to detect antibodies specific for common infectious agents such as Epstein–Barr virus (EBV), cytomegalovirus (CMV), Toxoplasma gondii (T. gondii), and hepatitis C virus (HCV) are time-consuming and require large volumes of samples, which restrict their use. We propose a new assay based on a multiplexed infectious protein (MIP) microarray combining different epitopes representative of the four germs. Antigens and lysates were printed on nitrocellulose slides to constitute the microarray. First, the microarray was incubated with human serum samples. Then, the suitability of the microarray for analysis of the specificity of purified monoclonal immunoglobulin (mc Ig) was assessed using serum and mc Ig of HCV-positive patients. Bound human immunoglobulin G (IgG) was detected using fluorescently labeled secondary antibodies, and the signals were quantified. Results obtained in serum samples with the new MIP microarray immunoassay were compared with ELI-SAs; we observed concordances of 95% for EBV, 93% for CMV, 91% for T. gondii, and 100% for HCV. Regarding purified mc Ig of HCV-positive patients, 3 of 3 recognized antigens printed on the microarray. Hence, the novel EBV/CMV/T. gondii/HCV MIP microarray allows simultaneous diagnosis of polyclonal and monoclonal immune response to infectious diseases using very small volume samples.