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[Display omitted] • Bacillus siamensis LZ88 showed significant antifungal activity against A. alternata. • Non-volatile and volatile organic compounds against A. alternata were identified. • LZ88 is a promising alternative to agrochemicals for biocontrol of A. alternata. Our previous study identified that Bacillus siamensis strain LZ88 has significant antagonistic activity against a variety of pathogenic fungi, especially against Alternaria alternata , which causes tobacco brown spot. In the present study, the non-volatile compounds and volatile organic compounds (VOCs) produced by LZ88 were identified and tested for their antifungal activities. The non-volatile compounds had significant inhibitory effects on both mycelial growth and spore germination of A. alternata , with IC 50 values of 24.25 μg/mL and 29.04 μg/mL, respectively. HPLC-DAD-ESI-MS/MS analysis revealed that the non-volatile compounds which showed antifungal activities were iturins and macrolactin. HS-SPME-GC–MS identified ten VOCs from strain LZ88, including 8 ketones and 2 acids. Among the VOCs tested, 2-methylbutanoic acid and 3-methylbutanoic acid exhibited significant antifungal activity against A. alternata. Both 2-methylbutanoic acid and 3-methylbutanoic acid could inhibit mycelium growth of A. alternata with IC 50 values of 83.10 mg/mL and 104.19 mg/mL, whereas inhibited spore germination with IC 50 values of 139.63 mg/mL and 88.07 mg/mL, respectively. Furthermore, when exposed to non-volatile compounds or VOCs, the hyphae and spores of A. alternata were deformed and collapsed, and consequent hyphae without any attached conidia appeared ruptured, shrunken, and twisted. Additionally, the genetic mechanisms were analyzed by whole genome sequencing, and the gene clusters responsible for antibacterial metabolites were mined. These results indicate that strain LZ88 possessed a remarkable potential as alternative agrochemicals toward A. alternata. [ABSTRACT FROM AUTHOR]