Background/Objectives: Sweetpotato black rot, caused by Ceratocystis fimbriata, is a severe fungal disease in sweetpotato production. Biological control strategies represent a promising, environmentally sustainable approach to managing this disease. This study investigates the biocontrol potential of Bacillus amyloliquefaciens SFB-1 against C. fimbriata. Methods: The antagonistic activities of strain SFB-1 on C. fimbriata were assessed through in vitro assays, including evaluations of mycelial inhibition, spore germination, and mycelial morphology. Pathogenicity assays on harvested sweetpotato roots assessed lesion diameter and depth. A transcriptomic analysis of C. fimbriata exposed to strain SFB-1 was performed to explore the underlying antifungal mechanism of SFB-1 on C. fimbriata. The qRT-PCR was employed to validate the RNA-seq results. Results: In vitro assays demonstrated that strain SFB-1 inhibited C. fimbriata mycelial growth by up to 81.01%, caused mycelial swelling, and completely suppressed spore germination at 10⁸ CFU/mL. The cell-free supernatant of strain SFB-1 also suppressed C. fimbriata growth. Pathogenicity assays revealed that strain SFB-1 treatments reduced lesion diameter and depth on harvested sweetpotato roots by over 50% compared to untreated controls. Transcriptomic analysis of C. fimbriata treated with strain SFB-1 identified 1164 differentially expressed genes, with significant alterations in genes associated with cell wall integrity, cell membrane stability, spore germination, detoxification, and antioxidant responses. The qRT-PCR validation of 16 genes confirmed the consistency with the RNA-seq results. Conclusions: B. amyloliquefaciens SFB-1 demonstrates significant biocontrol efficacy against C. fimbriata through multiple mechanisms, positioning it as a promising solution for the sustainable management of sweetpotato black rot.