Sweetpotato, a vital global crop, cash, and fodder crop, faces significant threats from black rot disease caused by Ceratocystis fimbriata (C. fimbriata). Benzyl isothiocyanate (BITC) is a biologically active essential oil derived from cruciferous plants, widely used for food preservation and exhibiting antimicrobial and antitumor properties. In this study, BITC significantly inhibited C. fimbriata growth and induced cellular damage. Furthermore, through proteomics and bioinformatics analyses, mitochondrial peroxiredoxin PRX1 (PRX1) was identified as a potential target protein of BITC in C. fimbriata, confirming its role in regulating the response of BITC-treated C. fimbriata to oxidative damage. Finally, sweetpotato storage simulation experiments demonstrated that BITC effectively prevents and controls the growth of C. fimbriata during sweetpotato storage. These results provide the basis for using BITC to control sweetpotato black rot, offering insights into developing highly selective and low-negative-impact antisweetpotato black rot disease compounds, and presenting a new strategy for controlling C. fimbriata contamination in sweetpotatoes.