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<i>Pestalotiopsis</i> sp. is an important class of plant pathogenic fungi that can infect a variety of crops. We have proved the pathogenicity of <i>P. kenyana</i> on bayberry leaves and caused bayberry blight. Phenazine-1-carboxylic acid (PCA) has the characteristics of high efficiency, low toxicity, and environmental friendliness, which can prevent fungal diseases on a variety of crops. In this study, the effect of PCA on the morphological, physiological, and molecular characteristics of <i>P. kenyana</i> has been investigated, and the potential antifungal mechanism of PCA against <i>P. kenyana</i> was also explored. We applied PCA on <i>P. kenyana</i> in vitro and in vivo to determine its inhibitory effect on PCA. It was found that PCA was highly efficient against <i>P. kenyana,</i> with EC₅₀ around 2.32 μg/mL, and the in vivo effect was 57% at 14 μg/mL. The mechanism of PCA was preliminarily explored by transcriptomics technology. The results showed that after the treatment of PCA, 3613 differential genes were found, focusing on redox processes and various metabolic pathways. In addition, it can also cause mycelial development malformation, damage cell membranes, reduce mitochondrial membrane potential, and increase ROS levels. This result expanded the potential agricultural application of PCA and revealed the possible mechanism against <i>P. kenyana</i>.