Current regulatory protocols for assessing herbicide effects on plants rely heavily on the use of crops grown under controlled greenhouse conditions to indicate risks to wild vegetation. Guideline test protocols call for low levels of soil nutrients, approximately 3% organic matter (OM), to test the worst-case scenario for non-target species growing in poor soils. However, species sensitivity to herbicides may be affected by growing conditions, especially soil nutrient levels. The purpose of this study was to assess the impact of high and low soil OM content on the sensitivity of plants to several common agricultural herbicides (glyphosate, chlorimuron ethyl and dicamba). Ten plant species (wild and crop) with high (nitrophilous) or low (non-nitrophilous) affinity for nitrogen were grown under greenhouse conditions in soil with two levels of OM (3 and 9%) and were exposed to seven doses of the three herbicides in concurrent experiments. Results showed that most species were more sensitive to glyphosate under high OM conditions while chlorimuron ethyl and dicamba results were mixed with respect to OM levels. Overall, 15 species-herbicide combinations were more sensitive in high OM soil, while 11 were more sensitive in low OM soil. No clear pattern in sensitivity emerged among nitrophilous and non-nitrophilous species. Several species showed a difference in response at herbicide levels expected to reach non-target habitats adjacent to crop fields through drift (5% commonly and 25% occasionally). In terms of regulatory testing, guidelines may need alteration to allow testing with nutrient levels that more accurately reflect natural environments.