Oncolytic adenoviral vectors hold promise as efficient anti-cancer therapy for the treatment of head and neck squamous cell carcinomas (HNSCC). Since the epidermal growth factor receptor (EGFR) has been shown to be overexpressed in many types of HNSCC, it represents an attractive target for oncolytic virotherapy. EGFR-specific targeting of human adenovirus type 5 (HAdV_5) -based vectors was achieved by a position-specific and covalent attachment of the EGFR affinity ligand Affilin to either the fiber or hexon protein of the vector capsid. In vitro, remarkably enhanced and EGFR-specific cancer cell transduction combined with reduced susceptibility to known sequestration mechanisms of HAdV-5 particles were shown for Affilin-decorated vectors. However, no improved transduction of EGFR-positive tumors was achieved in vivo, neither after systemic nor intratumoral vector administration. Follow-up analysis indicated this observation to be caused by a rapid vector particle consumption due to binding to murine EGFR, weak tumor vascularization and poor target receptor accessibility in the solid tumor. In conclusion, in vitro results demonstrated proof-of-concept that covalent attachment of a receptor-specific Affilin to the adenoviral capsid is an effective and versatile novel strategy to target cancer-specific receptors by adenoviral vectors. In view to EGFR, broad off-target transduction and hindered target accessibility within the solid tumor hampered efficient tumor transduction by Affilin-decorated vectors, challenging EGFR as a suitable target receptor for adenoviral vector-based tumor therapy.