Abstract BACKGROUND The natural compound heat‐stable antifungal factor (HSAF), produced by Lysobacter enzymogenes OH11, exhibits potent inhibitory activity against Fusarium graminearum and effectively controls Fusarium head blight, yet its mechanism remains unclear. Our previous work identified FGSG_09851, a microtubule‐severing protein containing a conserved AAA (ATPases associated with diverse cellular activities) domain, binding to HSAF in F. graminearum . Here, we investigated the biological function of FGSG_09851 and explored its potential as an antifungal target. RESULTS Deletion of FGSG_09851 led to a marked increase in HSAF resistance in F. graminearum (EC50: 1.602 ± 0.139 vs 0.787 ± 0.156 μg/mL for wild‐type), and impaired mycelial growth, conidial germination, conidiation, and virulence. The Δ FGSG_09851 mutant exhibited sparse and disorganized microtubules, consistent with that observed under HSAF treatment. Molecular docking showed that HSAF binds to the active pocket of FGSG_09851 with a binding energy of −7.9 kcal/mol. Using structural features of known AAA+ ATPase inhibitors, we designed three compounds, AH‐001, AH‐002, and AH‐003, which showed antifungal activity. Among them, AH‐001 was the most potent, with inhibition rates of 25.5%, 39.5%, and 47.8% against F. graminearum at 120, 150, and 180 μg/mL, respectively. At 180 μg/mL, AH‐002 and AH‐003 achieved 35.4% and 32.9% inhibition. CONCLUSION This study demonstrates that HSAF exerts its antifungal activity against F. graminearum by targeting the microtubule‐severing protein FGSG_09851. Guided by this mechanism, we designed and synthesized novel AAA+ ATPase inhibitors with promising antifungal activity. These findings elucidate the mode of action of HSAF and establish microtubule‐severing proteins as a promising target class for fungicide development. © 2026 Society of Chemical Industry.