Pfaffia glomerata is a medicinal plant widely distributed in Brazil, which is considered the world's greatest supplier of P. glomerata roots. Among active ingredients contained in this plant, the steroid β-ecydisone (20E) is the most important compound extracted from roots. This steroid presents therapeutic properties for the treatment of diabetes and haemorrhoids, besides having bioenergy, tonic and aphrodisiac effects. The root-knot nematode Meloidogyne spp. is a major limiting factor in root production. Recent studies showed resistance of accessions of P. glomerata to Meloidogyne incognita. The aims of this work were: i) to correlate the concentration of 20E with resistance and susceptibility of P. glomerata accessions to M. incognita in inoculated and non-inoculated plants; ii) to study the effect of the parasitism of M. incognita on the concentration of the steroid 20E in the roots; and iii) to clarify resistance mechanisms by comparing the response of a highly resistant UFV with a highly susceptible accession (Farm) to nematode infection. The concentration of 20E in the healthy susceptible Farm accession was significantly higher than in the healthy resistant UFV accession, showing that the resistance mechanism was not related to 20E concentrations. Plants of the Farm accession infected with M. incognita showed higher levels of 20E than the non-infected control. A positive and significant Pearson correlation coefficient was observed between 20E concentrations and gall indexes. Resistance of UFV to the root-knot nematode M. incognita was associated with unidentified factors that limited nematode penetration or emigration of second-stage juveniles and with post-penetration responses, including the hypersensitive response. Giant cells were sometimes found in the resistant cultivar, but displayed a highly vacuolated and degraded cytoplasm with thinner cell walls than those induced in the susceptible accession. Microscope observations under UV light showed a strong autofluorescence, suggesting that phenolic compounds may be involved in ginseng UFV resistance.