Infections caused by root-knot nematodes (RKNs) significantly impair vegetable growth and crop yield, posing a severe threat to global food security. Our previous study indicated that fungal-derived 2-furoic acid was a promising lead compound for the exploitation of eco-friendly nematicides. However, the exact molecular mechanism remains poorly understood. In this study, we observed behavioral changes in the nematodes following treatment with 100 μg/mL of 2-furoic acid. Subsequently, transcriptomics and metabolomics were combined to identify changes in differentially expressed genes (DEGs) and differentially expressed metabolites (DEMs). Transcriptome analysis revealed 199 up-regulated and 109 down-regulated DEGs. Metabolomics analysis indicated that 140 metabolites (72 up-regulated and 68 down-regulated) exhibited significant differences. Notably, the integrated transcriptomics and metabolomics analysis identified a total of 72 DEGs and 54 DEMs annotated across 51 pathways, including lipid metabolism and amino acid metabolism. Intriguingly, 14 out of the 51 pathways were simultaneously annotated to the downregulated calmodulin gene (cam). RNA interference (RNAi) results demonstrated that the down-regulated cam, a potential drug target for controlling RKNs, significantly reduced the motility, infectivity, and pathogenicity of M. incognita. Our results provide insights into the molecular basis of 2-furoic acid against M. incognita and offer a valuable theoretical foundation for the design of eco-friendly nematicides.