NV1066 is a herpes simplex virus-1 (HSV-1) oncolytic mutant that contains the gene for enhanced green fluorescent protein (EGFP). We sought to determine (1) whether NV1066 is effective against human peritoneal cancer, (2) whether EGFP is detectable in an animal model of gastric cancer, and (3) whether EGFP expression can be used to assess oncolytic therapy in a minimally invasive, laparoscopic system. The current study demonstrates that NV1066 is cytotoxic to OCUM human gastric cancer cells in vitro and in an in vivo model of disseminated peritoneal gastric cancer. In vitro this human gastric cancer cell line is sensitive to NV1066. Lysis occurs in a dose-dependent fashion, achieving near-complete lysis even at multiplicities of infection (MOIs) as low as 0.01 by 7 days. NV1066 also replicates within OCUM cells and induces expression of GFP in a dose-dependent manner. At MOIs of 0.01 to 1, EGFP expression is seen by flow cytometry in 100% of OCUM cells within 5 days after infection. NV1066 effectively treats OCUM carcinomatosis in an in vivo model. After intraperitoneal administration of NV1066, macroscopic tumor foci express EGFP by direct laparoscopy with the appropriate fluorescent filtering. Noncancerous organs are not infected and do not express EGFP. We conclude that NV1066 has significant oncolytic activity in vitro and in vivo and reliably induces EGFP expression in infected tumor cells. Furthermore, EGFP expression in intraperitoneal tumors can be visualized laparoscopically, allowing detection and localization of viral gene therapy.