The biocontrol agent Pythium oligandrum, which is a member of the phylum Oomycota, can control diseases caused by a taxonomically wide range of plant pathogens, including fungi, bacteria, and oomycetes. However, whether P. oligandrum could control diseases caused by plant root-knot nematodes (RKNs) was unknown. We investigated a recently isolated P. oligandrum strain GAQ1, and the P. oligandrum strain CBS530.74, for the control of an RKN Meloidogyne incognita infection of tomato (Solanum lycopersicum L.). Initially, P. oligandrum culture filtrates were found to be lethal to M. incognita second-stage juveniles (J2s) with up to 84% mortality 24 h after treatment compared to 14% in the control group. Consistent with the lethality to M. incognita J2s, tomato roots treated with P. oligandrum culture filtrates reduced their attraction of nematodes, and the number of nematodes penetrating the roots was reduced by up to 78%. In a greenhouse pot trial, the P. oligandrum GAQ1 inoculation of tomato plants significantly reduced the gall number by 58% in plants infected with M. incognita. Notably, the P. oligandrum GAQ1 mycelial treatment significantly increased tomato plant height (by 36%), weight (by 27%), and root weight (by 48%). A transcriptome analysis of tomato seedling roots inoculated with the P. oligandrum GAQ1 strain identified ~2500 differentially expressed genes. The enriched GO terms and annotations in the up-regulated genes suggested a modulation of the plant hormone-signaling and defense-related pathways in response to P. oligandrum. In conclusion, our results support that P. oligandrum GAQ1 can serve as a potential biocontrol agent for M. incognita control in tomato. Multiple mechanisms appear to contribute to the biocontrol effect, including the direct inhibition of M. incognita, the potential priming of tomato plant defenses, and plant growth promotion.