Two new antibiotics, neothramycins A and B, were isolated from the culture broth of Streptomyces No. MC916-C4 (a group C strain of cycloheximide-producing Streptomyces). They were produced in a specific aerated medium at 28°C for 4 days, then purified via activated carbon adsorption, n-butanol extraction, Sephadex LH-20 column chromatography (methanol as eluent), and silica gel column chromatography (chloroform-ethanol as eluent), with purification steps conducted at 5°C due to their lability. Their physicochemical properties were characterized: neothramycin A is a colorless amorphous powder (mp 132-147°C dec., [α]D +272° in dioxane) with molecular formula C₁₃H₁₄N₂O₄·1/2H₂O, and neothramycin B is similar (mp 144-151°C dec., [α]D +314° in dioxane) with the same molecular formula. UV, IR, PMR, and MS spectra were recorded, showing similarities and subtle differences (e.g., PMR signal of a methine proton) suggesting configurational differences at a methine carbon. Structural analysis revealed they are interconvertible isomers belonging to the anthramycin group antibiotics with a benzodiazepine structure, distinguishable from related antibiotics (anthramycin, dextrochrysin, sibiromycin, tomaymycins) by spectral or molecular formula differences. Biological activity tests showed weak antimicrobial activity against some bacteria (e.g., Staphylococcus aureus SMITH, Klebsiella pneumoniae PCI602) and fungi, but significant antitumor activity: they prolonged the survival period of mice with L-1210 leukemia (up to 200% prolongation at 150 mcg/mouse/day for 10 days) and Ehrlich ascites carcinoma, inhibited Yoshida rat sarcoma cells and SV40-transformed C3H cells in tissue culture. Acute LD50 in mice was 20-30 mg/kg via intravenous or intraperitoneal injection.