In our study to discover antitumor antibiotics from microorganisms, ansamycin antibiotics trienomycins A (1), B (2), and C (3) have been found in the culture broth of Streptomyces sp. No. 83-16 as strong cytotoxic compounds against HeLa S3 cells. This study was undertaken to isolate further minor components from the broth, and trienomycins D (4) and E (5) were consequently isolated. The fermentation process was carried out with specific media and conditions, followed by isolation via solvent extraction, Diaion HP-20 and HP-20SS column chromatography, and preparative HPLC. Structural analyses showed that trienomycin D (4) is a colorless powder decomposing at 137~139°C, with a UV spectrum very similar to that of trienomycin A, a molecular formula of C36H49N2O7 (elucidated by high resolution mass spectrometry), and a 13C NMR spectrum quite similar to that of trienomycin A except for the signal attributed to the acyl group attached to the alanyl moiety. Trienomycin E (5) was isolated using the same method.