Summary Endoplasmic reticulum (ER) bodies are ER‐derived structures that contain a large amount of PYK10 myrosinase, which hydrolyzes tryptophan (Trp)‐derived indole glucosinolates (IGs). Given the well‐described role of IGs in root–microbe interactions, we hypothesized that ER bodies in roots are important for interaction with soil‐borne microbes at the root–soil interface. We used mutants impaired in ER bodies (nai1), ER body‐resident myrosinases (pyk10bglu21), IG biosynthesis (myb34/51/122), and Trp specialized metabolism (cyp79b2b3) to profile their root microbiota community in natural soil, evaluate the impact of axenically collected root exudates on soil or synthetic microbial communities, and test their response to fungal endophytes in a mono‐association setup. Tested mutants exhibited altered bacterial and fungal communities in rhizoplane and endosphere, respectively. Natural soils and bacterial synthetic communities treated with mutant root exudates exhibited distinctive microbial profiles from those treated with wild‐type (WT) exudates. Most tested endophytes severely restricted the growth of cyp79b2b3, a part of which also impaired the growth of pyk10bglu21. Our results suggest that root ER bodies and their resident myrosinases modulate the profile of root‐secreted metabolites and thereby influence root–microbiota interactions.