Analyses of metabolite production and accumulation in roots of canola ( Brassica napus L. spp. oleifera) infected with the phytopathogen Plasmodiophora brassicae (clubroot) allowed the identification of 45 metabolites. HPLC analysis corroborated by metabolite isolation and NMR spectroscopic data demonstrated for the first time that phytoalexins and phytoanticipins were produced in roots of canola infected with a soilborne biotroph. In addition, six new indolyl metabolites were identified, synthesized, and tested against three fungal pathogens of canola. Multivariate data analysis using principal component analysis (PCA) revealed distinct metabolic responses of canola to P. brassicae infection during a six-week period. At late harvest days (five and six weeks), a clear clustering was observed among samples of infected roots because of the higher concentration of phytoalexins, while higher concentration of phytoanticipins contributed to the differentiation between three and four weeks samples of infected and control roots. Altogether, the data shows that canola roots under biotrophic attack are able to produce a complex blend of phytoalexins and other antimicrobial metabolites as a defensive response and that the metabolic regulation of phytoanticipins and phytoalexins appeared to correlate with the infection period.