In this study, a multiplex bead-based capture sandwich immunoassay (MIA) for the simultaneous detection of fifteen plant and bacterial Gram-positive and Gram-negative pathogenic biological agents (PBAs) was developed. This study included the selection of “sandwich” pairs of monoclonal antibodies, the development of an analytical protocol, and an evaluation of the specificity and sensitivity of detection. The working range, linearity, precision, and limits of detection of the developed assay were evaluated. The LLOQ of the multiplex assay was around 104 – 105 CFU/ml for bacteria and 0,4 ng/ml – 6,6 ng/ml for antigens. The assay can be used for the simultaneous detection of 15 PBAs, including Francisella tularensis, Brucella abortus, Bacillus anthracis, Legionella pneumophila, Staphylococcus aureus, Staphylococcus haemolyticus, Pseudomonas aeruginosa, and Salmonella typhimurium, as well as cholera toxin beta-subunit, viscumin, ricin, staphylococcal enterotoxins A and B, and botulinum toxins A and B.