Single-chain formats of TNF-related apoptosis inducing ligand (scTRAIL) can serve as effector components of tumour-associated antigen-targeted as well as non-targeted fusion proteins, being characterized by high tumour cell-specific induction of apoptosis through death receptor activation. We studied the suitability of immunoglobulin G as a scaffold for oligovalent and bispecific TRAIL fusion proteins. Thus, we developed novel targeted hexa- and dodecavalent IgG-scTRAIL molecules by fusing scTRAIL to the C-terminus of either light (LC-scTRAIL) or heavy immunoglobulin chain (HC-scTRAIL), or to both ends (LC/HC-scTRAIL) of the anti-EGFR IgG antibody hu225. The binding specificity to EGFR and death receptors was retained in all IgG-scTRAIL formats and translated into high antigen-specific bioactivity on EGFR-positive Colo205, HCT116 and WM1366 tumour cell lines, with or without sensitization to apoptosis by bortezomib. In vivo, therapeutic potential was assessed for one of the targeted variants, HC-scTRAIL, compared to the non-targeted Fc-scTRAIL. Both molecules showed a significant reduction of tumour volume and synergism with a Smac mimetic in a Colo205 xenograft tumour model. The IgG-scTRAIL format allows directing a defined, highly bioactive form of TRAIL to a wide variety of tumour antigens, enabling customized solutions for a patient-specific targeted cancer therapy with a reduced risk of side effects.