FcγRIII (CD16) plays an important role in the anti-tumor effects of therapeutic antibodies. Bi-specific antibodies (bsAbs) targeting FcγRIII represent a powerful alternative to the recruitment of the receptor via the Fc fragment, but are not efficiently produced. Single-domain antibodies (sdAbs) endowed with many valuable structural features might help to bypass this problem. In the present work, we have isolated anti-FcγRIII sdAbs (C21 and C28) from a phage library generated from a llama immunized with FcγRIIIB extra-cellular domains. These sdAbs bind FcγRIIIAsup+ NK cells and FcγRIIIBsup+ polymorphonuclear cells, but not FcγRIsup+ or FcγRIIsup+ cells, as detected by indirect immunofluorescence. Competition experiments showed that C21 and C28 sdAbs bind different FcγRIII epitopes, with C21 recognizing a linear and C28 a conformational epitope of the receptor. Surface plasmon resonance experiments showed that C21 and C28 sdAbs bind FcγRIII with a KsubD in the 10 and 80 nM range, respectively. Importantly, the engagement by both molecules of FcγRIIIA expressed by transfected Jurkat T cells or by NK cells derived from peripheral blood induced a strong IL-2 and IFN-γ production, respectively. These anti-FcγRIII sdAbs represent versatile tools for generating bsAbs under various formats, able to recruit FcγRIII killer cells to target and destroy tumor cells.