High bacterial density and diversity near plant roots has been attributed to rhizodeposit compounds that serve as both energy sources and signal molecules. However, it is unclear if and how specific rhizodeposit compounds influence bacterial diversity. We silenced the biosynthesis of isoflavonoids, a major component of soybean rhizodeposits, using RNA interference in hairy-root composite plants, and examined changes in rhizosphere bacteriome diversity. We used successive sonication to isolate soil fractions from different rhizosphere zones at two different time points and analyzed denaturing gradient gel electrophoresis profiles of 16S ribosomal RNA gene amplicons. Extensive diversity analysis of the resulting spatio temporal profiles of soybean bacterial communities indicated that, indeed, isoflavonoids significantly influenced soybean rhizosphere bacterial diversity. Our results also suggested a temporal gradient effect of rhizodeposit isoflavonoids on the rhizosphere. However, the hairy-root transformation process itself significantly altered rhizosphere bacterial diversity, necessitating appropriate additional controls. Gene silencing in hairy-root composite plants combined with successive sonication is a useful tool to determine the spatio temporal effect of specific rhizodeposit compounds on rhizosphere microbial communities.