Recent studies revealed that plant–soil biotic interactions may cause changes in above-ground plant chemistry. It would be a new step in below-ground–above-ground interaction research if such above-ground chemistry changes could be efficiently detected. Here we test how hyperspectral reflectance may be used to study such plant–soil biotic interactions in a nondestructive and rapid way. The native plant species Jacobaea vulgaris and Jacobaea erucifolius, and the exotic invader Senecio inaequidens were grown in different soil biotic conditions. Biomass, chemical content and shoot reflectance between 400 and 2500 nm wavelengths were determined. The data were analysed with multivariate statistics. Exposing the plants to soil biota enhanced the content of defence compounds. The highest increase (400%) was observed for the exotic invader S. inaequidens. Chemical and spectral data enabled plant species to be classified with an accuracy > 85%. Plants grown in different soil conditions were classified with 50–60% correctness. Our data suggest that soil microorganisms can affect plant chemistry and spectral reflectance. Further studies should test the potential to study plant–soil biotic interactions in the field. Such techniques could help to monitor, among other things, where invasive exotic plant species develop biotic resistance or the development of hotspots of crop soil diseases.