Previous results had documented oncolytic capacity of reovirus, parvovirus and Newcastle disease virus (NDV) on several tumor cell types. To test whether combinations of these viruses may increase this capacity, human U87- and U373-glioblastoma cells, in vitro or xenografted into immuno-compromised mice, were subjected to simultaneous double infections and analyzed. Our results show that reovirus (serotype-3) plus NDV (Hitcher-B1) and reovirus plus parvovirus-H1 lead to a significant increase in tumor cell killing in vitro in both cell lines (Kruskal-Wallis test, P < 0.01) and in vivo. Immunofluorescence and flow cytometry analyses demonstrated the simultaneous replication of the viruses in nearly all cells (>95%) after combined infection. These data thus indicate that a synergistic anti-tumor effect can be achieved by the combined infection with oncolytic viruses.