Ansamycin antibiotics, derived from 3-amino-5-hydroxybenzoic acid (AHBA), exhibit extensive biological activities such as antitumor, antiviral, and antibacterial. C17-benzene ansamycins (C17BAs), characterized by a 21-membered macrolactam linked to AHBA and a carboxylic acid moiety via L-alanine, have bioactive diversity dependent on the C-11 acyl chain and aromatic ring. Previously, four known C17BAs (trienomycin A (1), benzoxazomycin (2), mycotrienin II (3), mycotrienin I (4)) were isolated from Streptomyces seoulensis IFB-A01, and the conversion of 1 to 2 and its biosynthetic gene cluster were clarified. A mutant strain IFB-A01-C, with deleted genes for cyclohexanecarboxylic acid (CHC) biosynthesis, lost the ability to produce 1–4 but produced new analogs 1a–1c (major HPLC peaks), which were also present in the wild-type at ~10–20 times lower yields. Scaleup fermentation of the mutant enabled isolation, structural elucidation, and bioactivity assay of these analogs. Compounds 1a (trienomycin B) and 1b (trienomycin E) differed from 1 in the C-11 side chain, while 1c was a new derivative with an isobutyl group. Diene analogs 2a–2c were produced via enzymatic monooxygenation and spontaneous reactions, with 2a identified as a new diene ansamycin. Cytotoxicity assays showed 1b and 1c had stronger activity against HepG2 and MCF-7 than 1, with 1b comparable to doxorubicin. Anti-inflammatory assays revealed 2a suppressed LPS-induced IL-6 production in RAW264.7 more potently than 2. This study demonstrates that mutasynthesis combined with primary metabolism is an effective approach for mining new bioactive natural products from Streptomyces strains, which is valuable for drug discovery.