Background <p>Brucellosis, a severe zoonotic infectious disease, poses substantial economic and health threats globally. The intracellular survival strategy of <i>Brucella</i> complicates disease control, highlighting the need for novel immunotherapeutic strategies such as antibody-based therapies and multi-epitope vaccines.</p> Results <p>This study generated two IgM monoclonal antibodies (D3 and F5) against the conserved outer membrane protein OMP16 of <i>Brucella</i> using hybridoma technology. Peptide scanning and Western blot identified their linear epitopes (D3: ⁷⁷TLSKQAQW⁸⁴; F5: ¹²⁰RDFLASRG¹²⁷), which are highly conserved among major <i>Brucella</i> species. Integrated approaches—including molecular docking, alanine-scanning mutagenesis, and dot-blot assays—revealed key residues at the epitope interface that form stable bonds with antibody complementarity-determining regions (CDRs). Functionally, both antibodies activated the complement system, with F5 exhibiting significant complement-dependent bacteriolytic activity in vitro. Furthermore, in the presence of complement, D3 and F5 enhanced macrophage-mediated opsonophagocytosis and intracellular killing of <i>Brucella abortus</i> A19. In a mouse infection model, passive immunization with either antibody significantly alleviated infection-induced weight loss and splenomegaly and reduced bacterial load in the spleen.</p> Conclusions <p>Our study underscores the role of IgM antibodies in combating <i>Brucella</i> infection, offers insights for antibody-based immunotherapy, and provides a theoretical foundation for developing multi-epitope vaccines based on the conserved epitopes and critical residues.</p>