Targeted alpha-particle therapy (TAT) might be a relevant therapeutic strategy to circumvent resistance to conventional therapies in the case of HER2-positive metastatic cancer. Single-domain antibody fragments (sdAb) are promising vehicles for TAT due to their excellent in vivo properties, high target affinity and fast clearance kinetics. This study combines the cytotoxic α-particle emitter bismuth-213 (213Bi) and HER2-targeting sdAbs. The in vitro specificity, affinity and cytotoxic potency of the radiolabeled complex was analyzed on HER2pos cells. Its in vivo biodistribution through serial dissections and via Cherenkov and Micro-SPECT/CT imaging was evaluated. Finally, the therapeutic efficacy and potential associated toxicity of [213Bi]Bi-DTPA-2Rs15d was evaluated in a HER2pos tumor model that manifests peritoneal metastasis. In vitro, [213Bi]Bi-DTPA-2Rs15d bound HER2pos cells in a HER2-specific way. In mice, high tumor uptake was reached already 15 min after injection and extremely low uptake values were observed in normal tissues. Coinfusion of gelofusine resulted in a 2-fold reduction in kidney uptake. Administration of [213Bi]Bi-DTPA-2Rs15d alone and in combination with trastuzumab resulted in a significant increase in median survival. We describe for the very first time the successful labeling of an HER2-sdAb with the α-emitter 213Bi, and after i.v. administration, revealing high in vivo stability and specific accumulation in target tissue, and resulting in an increased median survival of these mice especially in combination with trastuzumab. These results indicate the potential of [213Bi]Bi-DTPA-sdAb as a new radioconjugate for TAT, alone and as an add-on to trastuzumab for the treatment of HER2pos metastatic cancer.