Background <p>Understanding how biostimulants modulate plant-associated microbiomes is critical for advancing sustainable agriculture. Here, we investigated the effects of <i>Bacillus</i>-derived bacillin 20 on the root and rhizosphere microbiomes of soybean (<i>Glycine max</i>) using amplicon-based profiling, community ecology, and network analysis.</p> Results <p>Microbial community assembly was driven primarily by plant compartmentalization, with higher bacterial richness in the rhizosphere and stronger host filtering in root-associated microbiomes. PERMANOVA analysis indicated that compartment explained most of the variation in community structure, whereas bacillin 20 treatment had no statistically significant effect. Despite no meaningful shifts in alpha diversity, bacillin 20 was associated with subtle, non-significant compositional trends in specific taxa across treatments. Indicator species and core microbiome analyses revealed compartment-specific taxa with potential roles in nutrient cycling, stress tolerance, and plant growth promotion.</p> Conclusion <p>Bacillin 20 was associated with changes in cross-domain microbial co-occurrence patterns, including differences in network connectivity, particularly in the rhizosphere, where several fungal ASVs (e.g., ASV33, ASV5, ASV8, and ASV88) exhibited high centrality. These findings indicate that bacillin 20 is associated with changes in microbial interaction patterns while maintaining overall community diversity. Overall, treatment effects were minor relative to compartment-driven structuring of the microbiome. Together, our results suggest that microbiome-informed approaches, including the use of targeted biostimulants, may contribute to the management of plant-microbe interactions in agricultural systems. Future studies integrating multi-omics approaches will be required to elucidate the underlying mechanisms of these interactions.</p>