Most terrestrial plants form mycorrhizas, but a number of agricultural plants, including the Brassicaceae, are non-mycorrhizal. Brassicaceae can still be colonized by arbuscular mycorrhizal fungi (AMF), but species like Arabidopsis thaliana experience growth reductions following AMF colonization at similar magnitude to that of fungal pathogen infections and lack key genes necessary for nutrient exchange. Arabidopsis also produces specific secondary compounds via the modification of tryptophan, including indolic glucosinolates (IGs), which have anti-fungal properties and may therefore be involved in reducing AMF colonization. This study therefore addressed whether the ability to produce IGs facilitates resistance to AMF colonization and growth suppression. We challenged with AMF inoculation transgenic Arabidopsis lines which produce no or enhanced IGs levels in comparison with the wild-type. Arbuscular mycorrhizal fungal inoculation suppressed the development of IG-removed plants, activated their pathogen-response defenses, and enhanced AMF vesicle colonization of their root systems. Arbuscular mycorrhizal fungi had no detrimental effects on wild-type or IG-enhanced plants. Using BLAST to identify IG orthologs across 29 Brassicales, we also show that non-mycorrhizal species possess orthologous proteins for IG biosynthesis to Arabidopsis which AMF-associated Brassicales lack. In conclusion, the IG production pathway appears to serve an important and previously unknown role in reducing AMF colonization in Arabidopsis and may serve similar functions in non-host Brassicales more broadly.