Exotic weeds can interfere with neighboring species by releasing allelochemicals that either directly inhibit growth and distribution of associated species or affect them indirectly by disrupting their interaction with soil organisms, such as arbuscular mycorrhizal fungi (AMF). The allelopathic potential of tall hedge mustard was assessed using aqueous root and shoot extracts in seed germination and radicle growth bioassays. Aqueous tall hedge mustard root and shoot extracts strongly inhibited seed germination and growth of bluebunch wheatgrass, Idaho fescue, and spotted knapweed, but had minimal autotoxicity. Chemical analysis of tall hedge mustard tissues revealed the presence of two major glucosinolates—isopropyl and sec-butyl glucosinolate. The degradation products of these glucosinolates (isopropyl isothiocyanate and sec-butyl isothiocyanate) were identified in dichloromethane extracts of tall hedge mustard aqueous root and shoot extracts. Commercially available isopropyl isothiocyanate and sec-butyl isothiocyanate inhibited seed germination and radicle growth, suggesting their role in the allelopathic potential of tall hedge mustard. Tall hedge mustard aqueous extracts and isothiocyanates incorporated into an agar medium inhibited the spore germination of the AMF, Glomus intraradices. Tall hedge mustard infestations were also found to reduce the AMF inoculum potential of soil under field conditions. The results from this study show that tall hedge mustard produces chemicals that can inhibit the growth of neighboring plant species and their AMF associates.