Bitetracenomycin A (1) and its diastereomers [(±)-bitetracenomycin B, (±)-2] were discovered from the cultures of Streptomyces sp. HDN154193. Compounds 1 and (±)-2 were the first tetracenomycin dimers obtained from a natural source with sp3 methine protons at the bridge positions (C-12/12'), which also exhibited broad-spectrum antibacterial activity. The racemate (±)-2 was semisynthesized and separated into enantiomers (+)-2 and (-)-2, and the absolute configurations were determined by specific rotation and ECD data. These metabolites exhibited potent antibacterial activity especially against drug-resistant strains (MRSA and MRCNS) with MIC values ranging from 1.0 to 1.9 μg/mL.