AbstractBACKGROUNDVerticillium wilt, caused by Verticillium dahliae, is a devastating soil‐borne disease. Spent mushroom substrate (SMS) has shown potential as a soil amendment for controlling soil‐borne diseases. However, the mechanisms underlying its disease‐suppressive effects remain poorly understood. Here, the efficacy of SMS in suppressing eggplant Verticillium wilt and mechanisms related to rhizosphere microbiome regulation were investigated.RESULTSWe tested different SMS sources (Pleurotus ostreatus, Hypsizygus marmoreus, Lentinus edodes), particle sizes (45, 75, 150, 300 μm), and addition ratios (0.5–8%, w/w). The fungus control efficacy ranged from 26 to 66%, with best results from 2% SMS of L. edodes at 150 μm. This treatment resulted in 5.7‐fold reduction in the Verticillium dahliae population in eggplant rhizosphere. Eggplant fresh and dry weights of shoots and roots, and plant height, significantly increased with 2% SMS amendment. 16S rDNA sequencing revealed alterations in rhizosphere bacterial communities, with an increase in indigenous beneficial bacteria, particularly Bacillus spp., following SMS amendment. Spent mushroom substrate co‐inoculated with exogenous biocontrol strain Bacillus subtilis NCD‐2 achieved a synergistic effect against Verticillium wilt than both SMS or NCD‐2 alone.CONCLUSIONSResults revealed that SMS protects eggplants against Verticillium wilt, largely by recruiting Bacillus spp. to the rhizosphere. The enrichment effect of indigenous Bacillus spp. in the rhizosphere mediated by SMS similarly applies to Bacillus inoculum, enhancing its efficacy in controlling eggplant Verticillium wilt. These findings enhance our understanding of the protective effects of SMS and its role in the biocontrol of Verticillium wilt. © 2025 Society of Chemical Industry.