Ansamycin antibiotics, derived from 3-amino-5-hydroxybenzoic acid (AHBA), exhibit antitumor, antiviral and antibacterial activities. C17-benzene ansamycins (C17BAs) possess a 21-membered macrolactam and a carboxylic acid moiety attached via L-alanine to the polyketide backbone, with bioactive diversity depending on the C-11 acyl chain and aromatic ring. During screening for new C17BA analogs, four known compounds (trienomycin A (1), benzoxazomycin (2), mycotrienin II (3), mycotrienin I (4)) were isolated from wild-type Streptomyces seoulensis IFB-A01, and previous work clarified the conversion from 1 to 2 and the biosynthetic gene cluster of 1, laying the foundation for mutasynthesis. A mutant strain (IFB-A01-C) with deleted genes related to cyclohexanecarboxylic acid (CHC) biosynthesis lost the ability to produce 1–4 but produced three new analogs (1a–1c), which were also detected in the wild-type strain at 10–20 times lower yields. Scale-up fermentation of the mutant strain was performed for isolation, structural elucidation and bioactivity assay. 1a and 1b were identified as trienomycin B and E, respectively, while 1c was a new derivative with an isobutyl group at the alanine moiety. Diene-type analogs 2a and 2c were also obtained. Cytotoxic assays showed 1b and 1c had stronger cytotoxicity against HepG2 (human hepatic carcinoma) and MCF-7 (human breast cancer) cells than 1, with 1b showing activity against HepG2 comparable to doxorubicin. Anti-inflammatory assays revealed 2a suppressed lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) production in RAW264.7 macrophages more potently than 2 without cytotoxic effects. This study demonstrates that combining mutasynthesis with primary metabolism is an effective approach to mine new bioactive natural products, which is valuable for constructing diversified compound libraries and drug discovery.