Root-knot nematodes belonging to different species of Meloidogyne cause great yield losses in tomatoes in Brazil. Among the control measures against these nematodes, alternative strategies that will avoid or reduce the use of nematicides have been investigated. The use of mushroom substrate proved to be an option due to its in vitro effect against nematodes. As a consequence, the goals of this study were to determine whether the infection process of Meloidogyne javanica could be affected on the roots of tomato plants growing on soil mixed with substrate of mushrooms belonging to the species Lentinula edodes, Macrocybe titans, and Pleurotus eryngii as well as to assess the disease on the roots of tomato plants whose seedlings were produced in substrate made of mushroom. The mixture of soil with the mushroom substrate obtained from L. edodes (isolate Led01), L. edodes (isolate Led02), M. titans (isolate Mti01), and P. eryngii (isolate Per01) significantly reduced gall index by 52, 73, 60, and 30%, and the nematode reproduction factor by 55, 39, 47, and 35%, respectively. On opposite, the galls index and reproduction factor of M. javanica were not affected on the roots of tomato plants whose seedlings were produced in the mushroom substrate. In conclusion, using mushroom substrate to produce tomato plants is a great option to reduce the infection process of M. javanica on the roots of plants.