Malignant pleural mesothelioma (MPM) is a highly aggressive tumor that is related to asbestos exposure. MPM is characterized by rapid and diffuse local growth in the thoracic cavity, and it has a poor prognosis because it is often refractory to conventional therapy. Although MPM is an extraordinarily challenging disease to treat, locoregional virotherapy may be useful against this aggressive disease because of the accessibility by intrapleural virus delivery. In this study, we show that telomerase-specific, replication-selective adenovirus OBP-301 can efficiently infect and kill human mesothelioma cells by viral replication. Intrathoracic administration of virus significantly reduced the number and size of human mesothelioma tumors intrathoracically implanted into nu/nu mice. A high-definition, fluorescence optical imaging system with an ultra-thin, flexible fibered microprobe clearly detected intracellular replication of green fluorescent protein-expressing oncolytic virus in intrathoracically established mesothelioma tumors. As the extracellular matrix (ECM) may contribute to the physiological resistance of a solid tumor by preventing the penetration of therapeutic agents (including oncolytic viruses), we also examined whether the co-expression of heparanase, an endoglucuronidase capable of specifically degrading heparan sulfate, that influences the physiological barrier to macromolecule penetration, can modify the permeability of the ECM, resulting in profound therapeutic efficacy. Co-injection of OBP-301 and a replication-defective adenovirus (Ad-S/hep)-expressing heparanase resulted in more profound antitumor effects without apparent toxicity in an orthotopic pleural dissemination model. Our results suggest that intrathoracic dual virotherapy with telomerase-specific oncolytic adenovirus in combination with heparanase-expressing adenovirus may be efficacious in the prevention and treatment of pleural dissemination of human malignant mesothelioma.