Chemical defenses are thought to contribute to the invasion success and impacts of many introduced plants; however, for most of these species, little is known about these compounds and how they vary in natural environments. Plant allelochemical concentrations may be affected by a variety of abiotic and biotic factors, including soil nutrients and herbivores. Moreover, such quantitative variation is likely to play an important role in species interactions involving these invasive plants. The purpose of this study was to examine patterns of variation in iridoid glycoside concentrations of the invasive plant Linaria dalmatica (Plantaginaceae). We conducted a greenhouse experiment to investigate the effect of soil nitrogen availability on iridoid glycoside concentrations. Results from this experiment showed that plant iridoid glycoside concentrations decreased with increased nitrogen availability. Additionally, plants were collected from multiple field sites in order to characterize the influence of population, soil nitrogen availability, and herbivore attack on iridoid glycoside variation. Results from field studies indicated that plants demonstrated considerable seasonal variation, as well as variation within and among populations, with iridoid glycoside concentrations ranging from approximately 1 to 15% dry weight. The relationship between soil nitrogen and plant iridoid glycosides varied among populations, with a strong negative correlation in one population, a marginally significant negative relationship in a second population, and no relationship in the remaining two populations. Additionally, we found a negative relationship between iridoid glycoside concentrations and plant injury by an introduced biocontrol agent, the stem-mining weevil Mecinus janthinus (Cucurlionidae). These results show that plant allelochemical concentrations can vary widely in natural environments and suggest that levels of plant defense may be reduced by increased soil nitrogen availability and herbivore attack in this invasive plant species.