Isoprene is the most abundant biogenic volatile organic compound emitted by terrestrial vegetation. Here we report on isoprene’s effect on root-associated microbiomes. Using isoprene emitting (IE) transgenic tobacco and isogenic non-emitting (NE) controls, we performed co-cultivation experiments in natural soil and analyzed plant phenotypes and growth alongside bacterial and fungal communities across root, rhizosphere, and soil niches. NE plants co-cultivated with IE neighbors displayed increased shoot and root biomass, suggesting interactive belowground functions of isoprene. Amplicon sequencing revealed that IE plants had more growth-promoting microbiota in root and rhizosphere compared with NE plants. Both bacterial and fungal growth-promoting microbiota were enriched in IE and NE plants grown in the same pot. However, isoprene-fumigated plant-free soils did not replicate these shifts, indicating that plant–microbe interactions are required for the modulation of the soil microbiome positions. Our results reveal that isoprene acts as a belowground cue influencing microbiome assembly and indirectly enhancing growth in neighboring plants. This work uncovers a potential ecological role for isoprene, highlighting how plant-derived isoprene can mediate plant–plant–microbiome interactions and contribute to community-level processes in the rhizosphere.