Microorganisms produce a wealth of structurally diverse specialized metabolites with a remarkable range of biological activities. The Phomopsis sp. LGT‐5 was obtained through tissue block and repeatedly crossed methods from Tripterygium wilfordii Hook. F. The antibacterial experiments of LGT‐5 showed that it has high inhibitory activity against Staphylococcus aureus and Pseudomonas aeruginosa, and moderate inhibitory activity against Candida albicans. To research the generation of the antibacterial phenomenon of LGT‐5 and provide support for further research and application, the whole genome sequencing (WGS) of LGT‐5 was obtained by single‐molecule real‐time DNA sequencing platform Pacific Biosciences (PacBio) sequencing and Illumina paired‐end sequencing. The final assembled LGT‐5 genome is 54.79 Mb with a contig N50 of 290.07 kb; in addition, its secondary metabolites were detected through HPLC‐Q‐ToF‐MS/MS. By comparing its MS/MS data, the secondary metabolites were analyzed based on visual network maps obtained on the Global Natural Products Social Molecular Networking (GNPS). The analysis results showed that the secondary metabolites of LGT‐5 were triterpenes and various cyclic dipeptides.