Southern blight is a common and devastating disease adversely affecting the yield and quality of cultivated Dendrobium officinale plants. The pathogens responsible for this disease are Sclerotium delphinii and S. rolfsii, although S. delphinii is more pathogenic than S. rolfsii. Plant endophytes are potential sources of biological control agents. In this study, 18 microbial strains were isolated from wild Dendrobium shixingense plants (e.g., Streptomyces, Bacillus, and Trichoderma), among which strain MEPF0303 significantly inhibited S. delphinii mycelial growth. Based on 16S rRNA gene sequence, this strain was identified as Streptomyces sp. In addition, the volatile organic compounds and cell-free supernatant of MEPF0303 had significant inhibitory effects on S. delphinii growth. Moreover, MEPF0303 effectively controlled southern blight of D. officinale. A total of 1,797 differentially expressed genes were identified by a comparative transcriptome analysis of S. delphinii mycelia treated with MEPF0303 and the untreated control mycelia. Strain MEPF0303 significantly altered the expression of genes related to cell membrane morphological development and integrity, pathogenicity, energy metabolism, and oxidative stress responses. Furthermore, the transcriptome sequencing results were validated by analyzing gene expression via a quantitative real-time polymerase chain reaction. The study results showed that MEPF0303 can inhibit the growth and pathogenicity of S. delphinii, with potential implications for the biological control of southern blight of D. officinale.