A pineapple-banana rotation was studied as a model system to investigate the potential emergence of a fungal-mediated disease-suppression in a soil highly infested with the pathogen Fusarium oxysporum causing the banana wilt disease. By using both field and pot experiments, the pineapple-banana rotation system resulted in a significant decrease of the pathogen number and next-stubble banana disease incidence (P < 0.05). This pathogen-suppression phenomenon was linked with detectable shifts in the soil resident fungal taxa tracked in the pineapple season. Most importantly, taxa affiliated with Talaromyces pinophilus and Clonostachys rossmaniae were found to be significantly enriched in the bulk soils due to the pineapple cultivation (P < 0.05). The taxon T. pinophilus was also significantly enriched in the rhizosphere of banana after the rotation (P < 0.05). Later, we used fungal isolation and pot inoculation to validate that both T. pinophilus and C. rossmaniae taxa are able to significantly decrease the pathogen number in the banana rhizosphere soil (P < 0.05), thus confirming their biocontrol effects suppressing the disease. Taken together, this study provides evidence on how crop rotation affects the resident soil microbiome and the development of disease suppressiveness. Besides, this study highlights the importance of understanding the dynamic changes in soil biology mediated by crop rotation and validates the mechanisms underpinning suppression toward promoting practical and directed manipulation of protective microbiomes in agroecosystems.