Gray blight is a serious disease of tea (Camellia sinensis) for which there is currently no effective control or preventive measure apart from fungicides. Screening for effectiveness of a natural antimicrobial against this pathogen and identifying its mode of action could contribute to the management of this disease. Antifungal activity of the antimicrobial ningnanmycin (NNM) from Streptomyces noursei var. xichangensis against the pathogen causing gray blight disease, Pseudopestalotiopsis camelliae-sinensis strain GZHS-2017-010, was confirmed in vitro by the mycelial growth rate method. Optical microscopy, scanning electron microscopy, and transmission electron microscopy were used to observe morphological changes in hyphae of P. camelliae-sinensis treated with NNM. RNA sequencing, bioinformatics, and quantitative real-time PCR were used to identify genes in the hyphae that were differentially expressed in response to treatment with NNM. Thirty-eight genes from 16 pathways, known as targets of antifungal agents, were used to investigate gene expression in hyphae at the half-maximal effective concentration (EC50), EC30, and EC70 for 1, 7, or 14 h. The results indicated that NNM can inhibit the growth of hyphae in vitro, with an EC50 of 75.92 U/ml, inducing morphological changes in organelles, septa, and extracellular polysaccharides, targeting ribosomes to disturb translation in protein synthesis and influencing some biosynthetic functions of the hyphae.