In previous studies we found that uveal melanoma cells grown in extracellular matrix (ECM)-containing three-dimensional (3D) cultures have increased resistance against herpes simplex virus type 1 (HSV-1)-mediated destruction relative to cells cultured without ECM. Using additional tumor cell types including MB-231 human breast cancer cells, PC-3 human prostate cancer cells, and P19 mouse embryonal carcinoma cells, we show here that tumor cell lines other than melanoma are also more resistant to HSV-1-mediated destruction in 3D cultures than cells grown in 2D. We also demonstrate here that one mechanism responsible for the increased resistance of tumor cells to HSV-1 infection in 3D cultures is an ECM-mediated inhibition of virus replication following virus entry into cells. These findings confirm and extend previous observations related to the role of the ECM in tumor resistance against HSV-1 and may lead to improved strategies of oncolytic virotherapy.