AbstractAcinetobacter baumanniiis a Gram-negative bacterial pathogen that exhibits high intrinsic resistance to antimicrobials, with treatment often requiring the use of last-resort antibiotics. Antibiotic-resistant strains have become increasingly prevalent, underscoring a need for new therapeutic interventions. The aim of this study was to useA. baumanniiouter membrane vesicles as immunogens to generate single-domain antibodies (VHHs) against bacterial cell surface targets. Llama immunization with the outer membrane vesicle preparations from fourA. baumanniistrains (ATCC 19606, ATCC 17961, ATCC 17975, and LAC-4) elicited a strong heavy-chain IgG response, and VHHs were selected against cell surface and/or extracellular targets. For one VHH, OMV81, the target antigen was identified using a combination of gel electrophoresis, mass spectrometry, and binding studies. Using these techniques, OMV81 was shown to specifically recognize CsuA/B, a protein subunit of the Csu pilus, with an equilibrium dissociation constant of 17 nM. OMV81 specifically bound to intactA. baumanniicells, highlighting its potential use as a targeting agent. We anticipate the ability to generate antigen-specific antibodies against cell surfaceA. baumanniitargets could provide tools for further study and treatment of this pathogen.Key points•Llama immunization with bacterial OMV preparations for VHH generation•A. baumannii CsuA/B, a pilus subunit, identified by mass spectrometry as VHH target•High-affinity and specific VHH binding to CsuA/B and A. baumannii cellsSupplementary InformationThe online version contains supplementary material available at 10.1007/s00253-023-12594-1.