Novel combinatorial treatment strategies are desired to achieve tumor eradication. In this regard, nanotechnology and gene therapy hold the potential to expand the available tumor treatment options. In particular, gold nanoparticles (AuNPs) have been utilized for hyperthermic tumor cell ablation. Similarly, adenoviral (Ad) vectors have been utilized for targeting, imaging, and cancer gene therapy. Thus, to combine AuNP-mediated hyperthermia with Ad vector-based gene therapy, we have previously coupled AuNPs to Ad vectors. Herein we tested the capability of these AuNP-coupled Ad vectors for hyperthermic tumor cell ablation. Towards this end, we compared absorption characteristics of different sized AuNPs and determined that in our system 20 nm diameter AuNPs are suitable for laser induced hyperthermic tumor cell killing. In addition, we observed that AuNPs outside and inside the cell contribute differentially towards hyperthermia induction. Unfortunately, due to the limitation of delivery of required amounts of AuNPs to cells, we observed that AuNP-coupled Ad vectors are unable to kill tumor cells via hyperthermia. However, with future technological advances, it may become possible to realize the potential of the multifunctional AuNP-coupled Ad vector system for simultaneous targeting, imaging, and combined hyperthermia and gene therapy of tumors.