Malignant pleural mesothelioma is a highly aggressive tumor. Alternative treatment strategies such as oncolytic viral therapy may offer promising treatment options in the future. In this study, the oncolytic efficacy and induction of tumor remission by a genetically engineered Newcastle disease virus [NDV; NDV(F3aa)-GFP; GFP, green fluorescent protein] in malignant pleural mesothelioma is tested and monitored by bioluminescent tumor imaging. The efficacy of NDV(F3aa)-GFP was tested against several mesothelioma cell lines in vitro. Firefly luciferase–transduced MSTO-211H* orthotopic pleural mesothelioma tumor-bearing animals were treated with either single or multiple doses of NDV(F3aa)-GFP at different time points (days 1 and 10) after tumor implantation. Tumor burden was assessed by bioluminescence imaging. Mesothelioma cell lines exhibited dose-dependent susceptibility to NDV lysis in the following order of sensitivity: MSTO-211H > MSTO-211H* > H-2452 > VAMT > JMN. In vivo studies with MSTO-211H* cells showed complete response to viral therapy in 65% of the animals within 14 days after treatment initiation. Long-term survival in all of these animals was >50 days after tumor installation (control animals, <23 d). Multiple treatment compared with single treatment showed a significantly better response (P = 0.005). NDV seems to be an efficient viral oncolytic agent in the therapy of malignant pleural mesothelioma in an orthotopic pleural mesothelioma tumor model. Mol Cancer Ther; 9(10); 2761–9. ©2010 AACR.