The primary cause of postharvest loss in table grape fruit is attributed to gray mold, which is caused by Botrytis cinerea. The present study confirmed the inhibitory effects of nystatin on the growth and development of B. cinerea, which led to a remarkable reduction in the severity of gray mold on table grape fruits. Furthermore, the application of nystatin disrupted the membrane permeability of B. cinerea, causing increased cellular leakage and cell death. In addition, the transcriptome analysis showed that the application of nystatin effectively modulated the transcriptional profile of genes involved in ribosome and mitochondrion biogenesis, as well as oxidoreductase activity, thereby disrupting the homeostasis of cellular organelles. Moreover, the nystatin treatment down-regulated genes associated with membrane trafficking, protein degradation by the ubiquitin–proteasome system, and the autophagy process, ultimately attenuating the pathogenicity of B. cinerea. Collectively, nystatin can be considered a viable agent for managing gray mold on table grape fruit.