Bifunctional chelators (BFCs) are covalently linked to biologically active targeting molecules and radiolabeled with radiometals. Technetium-99 m (Tc) is the most widely used isotope in nuclear medicine because of its excellent physical properties. The objective of this study was to synthesize and characterize a novel BFC that allows for the labeling of antibodies and antibody fragments using the Tc(CO) core which forms a very stable complex with Tc in the +1 oxidation sate. This study reports the synthesis of a BFC 1-pyrrolidinyl-2,5-dione-11-(bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino)undecanoic acid (SAAC-CIM NHS ester), and the in vitro and in vivo evaluation of Tc(CO)-SAAC-CIM-DLO6-(scFv) (Tc(CO)-DLO6-(scFv)), a domain I/II-specific anti-epidermal growth factor receptor I (anti-EGFR) antibody fragment. The chelator allowed radiolabeling the (scFv) antibody fragment in very mild conditions with no significant decrease in binding to EGFR. Radiochemical yields of >50% (radiochemical purity > 95%) of the resulting anti-EGFR (scFv) immunoconjugate Tc(CO)-DLO6-(scFv) was obtained. The radioimmunoconjugate was stable in histidine challenge experiments with less than 20% transchelation at 24 h after challenge in the presence of a 1500-fold excess of histidine. In vivo biodistribution of Tc(CO)-DLO6-(scFv) indicates that the tracer was mainly cleared via renal excretion and to a lesser extent via the hepatobiliary pathway. The microSPECT imaging studies performed in mice confirmed the in vitro affinity results. The Tc(CO)-DLO6-(scFv) shows some promising properties and warrants further investigation for imaging EGFR.