Root-knot nematodes (RKNs) are microscopic roundworms that infect the root of more than 2000 plant species, including major crop species. RKN infection results in general nutritional deficiency symptoms that resulted in loss in crop yield and productivity. It is estimated that RKNs contributed to up to $USD80 billion loss globally each year. Flavonoids are plant secondary metabolites with diverse functions in UV protection, pigmentations, signalling, auxin transport modulation and defence. My thesis explores the role of flavonoids in protecting the plant against RKN. To do this, I have profiled 30 flavonoids in the plant, Medicago truncatula which had been infected with the RKN species, Meloidogyne javanica using liquid tandem mass spectrometry. These results demonstrated that isoflavonoids were elicited in high amounts in response to nematode infection. Subsequently, I generated transgenic M. truncatula plants which over- and under-produced isoflavonoids. Additionally, I have tested M. truncatula accessions and cultivars with varied susceptibilities to RKN infection for differences in flavonoid regulation. The different M. truncatula genotypes showed that the early production or very high accumulation of isoflavonoids resulted in less severe infection phenotypes. To ascertain the role of isoflavonoids in protecting the plants against nematodes, I have designed and optimised in vitro assays to test the bioactivities of isoflavonoids in altering nematode motility and chemotaxis. The isoflavonoid afromosin and an isoflavonoid derivative, medicarpin were effective in inhibiting nematode motility and in repelling nematodes. Altogether, these results showed that isoflavonoids were produced by the host to defend itself from nematode infection.