Plant trichomes are known for their capability to produce and store secondary metabolites that protect plants from biotic and abiotic stresses. (1)H NMR studies on intact individual trichomes located on the leaf surface of Nicotiana attenuata revealed the presence of two major secondary metabolites: nicotine, the signature metabolite of the genus, and phaseoloidin, a homogentisic acid glucoside. This glucoside was reported originally from the seeds of Entada phaseoloides, and this is the first report of its occurrence in a Solanaceous plant. Artificial diet feeding bioassays with Manduca sexta and Spodoptera littoralis larvae, two important herbivores of N. attenuata, revealed that the ingestion of phaseoloidin negatively influenced caterpillar performance. This effect was more pronounced for the generalist, S. littoralis, than for the specialists, M. sexta.