Batatasin-III (3,3-dihydroxy-5-methoxybibenzyl) is a phenolic compound associated with the allelopathic effect of the evergreen dwarf shrub Empetrum nigrum, and has been referred to as the causal factor for the species being successful in dominating extensive ecosystems. Yet, only a few plant species have been tested for their response to batatasin-III, and little is known about whether environmental factors modify this allelopathic effect. In this study, we tested the inhibitory effect of purified batatasin-III through bioassays on 24 vascular plant species and, for certain species, we tested if this effect depended on growth substrate (mineral vs organic substrate), pH, and fertilization. Moreover, we tested if batatasin-III predicted the allelopathic effect of E. nigrum by analyzing the inhibitory effect of E. nigrum leaves and humus in relation to their batatasin-III content. Our results confirmed batatasin-III as a stable compound capable of inhibiting germination and/or mean root elongation in all of the tested species, but this effect was modified by growth substrate. Surprisingly, the measured batatasin-III content of E. nigrum leaves (mean value 19.7 ± 10.8 (SE) mg g−1) and humus (mean value of 1 ± 1.5 (SE) μg g−1) did not predict the inhibitory effect on mean root elongation. Although batatasin-III was found to be phytotoxic to all the tested species, we conclude that this substance alone should not be used as a proxy for the allelopathic effect of E. nigrum.